Immunoreactive protein orthologs of ehrlichia canis and e. chaffeensis

ABSTRACT

The present invention concerns gp36 immunoreactive compositions for  E. canis  and gp 47 immunoreactive compositions for  E. chaffeensis . In particular, epitopes for  E. canis  gp36 and  E. chaffeensis  gp 47 are disclosed. In certain embodiments, the immunoreactive compositions comprise tandem repeats having carbohydrate moieties.

The present invention is a continuation of U.S. application Ser. No.14/072,029, filed Nov. 5, 2013, which is a divisional of U.S.application Ser. No. 11/917,390, now U.S. Pat. No. 8,591,906, filed as anational phase application under 35 U.S.C. 371 of International PatentApplication PCT/US2006/023397, filed Jun. 15, 2006, which claims benefitof and priority to U.S. Provisional Patent Application No. 60/691,058,filed Jun. 16, 2005, all of which are incorporated by reference hereinin their entirety.

FIELD OF THE INVENTION

The present invention concerns at least the fields of molecular biology,cell biology, pathology, and medicine, including veterinary medicine. Inspecific aspects, the present invention concerns immunoreactive gp47 andgp36 proteins in Ehrlichia chaffeensis and E. canis, respectively.

BACKGROUND OF THE INVENTION

Ehrlichiae are tick-transmitted, obligately intracellular gram-negativebacteria that primarily reside within cytoplasmic vacuoles (earlyendosomes) of professional phagocytes, including macrophages andneutrophils. E. canis causes canine monocytic ehrlichiosis (CME), aserious and sometimes fatal globally distributed disease (Troy andForrester, 1990). E. chaffeensis causes human monocytotropic ehrichiosis(HME) in the United States, an emerging life-threatening disease inhumans, and also causes mild to severe ehrlichiosis in canines(Breitschwerdt et al., 1998). The importance of E. canis as a veterinarypathogen in conjunction with the recent identification of E. chaffeensisas the cause of an emerging tick-borne zoonosis has highlighted the needfor improved diagnostics and vaccines for both veterinary and humanehrlichioses, and thus the need for identification of immunoreactiveproteins.

A small subset of proteins expressed by E. canis and E. chaffeensisreact strongly with antibodies and are considered to be majorimmunoreactive proteins (Chen et al., 1997; Chen et al., 1994; McBrideet al., 2003). Several of these proteins have been molecularlycharacterized, including a 200-, 140/120-, and the 28-kDa multigenefamily of proteins (McBride et al., 2000a; Ohashi et al. 2001; Ohashi etal., 1998a; Ohashi et al., 1998b; Reddy et al., 1998; Yu et al., 1997;Yu et al., 2000a; Yu et al., 2000b), all of which are glycoproteins(McBride et al., 2003; McBride et al., 2000; Singu et al., 2005). Untilrecently, bacteria were thought to be incapable of proteinglycosylation, but numerous glycoproteins have recently been identifiedin various pathogenic bacteria (both intracellular and extracellular),including Ehrlichia (Benz and Schmidt, 2002; McBride et al., 2003;Schmidt et al., 2003; Upreti et al., 2003). Glycoproteins in pathogenicbacteria that have been functionally characterized include adhesins,toxins, and proteins involved in structural stability or mobility(Upreti et al., 2003). Some bacterial glycoproteins are highlyimmunogenic, highlighting a potential role in the development ofprotective immunity (Benz and Schmidt, 2002).

Several glycoproteins have been identified in Ehrlichia spp., includingsurface exposed proteins. The E. chaffeensis gp120 and E. canis gp140are major immunoreactive surface protein orthologs that have repeatunits with high serine and threonine content, and are involved inehrlichial attachment to the host cell (Popov et al., 2000). The gp200orthologs are the largest major immunoreactive proteins of Ehrlichiaspp. and are found primarily in the ehrlichial cytoplasm (McBride etal., 2003). The p28/p30 multigene family of proteins comprise the majorconstituents of the outer membrane and are thought to play a role insurface antigenic diversity and perhaps immune evasion (Ohashi et al.,1998; Reddy et al., 1998; Yu et al., 2000). Glycosylation andphosphorylation of the p28/p30 proteins has been reported in E.chaffeensis (Singu et al., 2005). At least some protection in mice hasbeen observed after immunization with recombinant p28/p30 (Ohashi etal., 1998).

The differential expression of ehrlichial antigens in tick and mammaliancells has been reported (Singu et al., 2005). Ehrlichial antigensexpressed in the tick or expressed soon after inoculation in the hostare likely to be recognized earliest by the host immune response. Thekinetics of the antibody response that develops to the majorimmunoreactive proteins of E. canis has been investigated inexperimentally-infected dogs (McBride et al., 2003). Two proteins, ofapproximately 19- and 37-kDa, were found to elicit the earliest acutephase antibody response, while the antibody response to p28/p30 majorouter membrane proteins as well as others developed two weeks later. Atotal of eight major immunoreactive proteins were recognized byantibodies in convalescent sera six weeks after inoculation (McBride etal., 2003).

The present invention fulfills a need in the art by providing novelmethods and compositions concerning Erhlichial infections in mammals.

SUMMARY OF THE INVENTION

Canine monocytic ehrlichiosis is a globally-distributed tick-bornedisease caused by the obligate intracellular bacterium E. canis and is auseful model for understanding immune and pathogenic mechanisms of E.chaffeensis, the causative agent of human monocytotropic ehrlichiosis.In general, the present invention concerns ehrlichial immunogeniccompositions, including immunoprotective antigens as vaccines forehrlichial diseases, such as subunit vaccines, for example. Theimmunogenic composition may be employed for any mammal, including, forexample, humans, dogs, cats, horses, pigs, goats, or sheep.

In particular, the present invention concerns the identification of thethird pair of molecularly and antigenically divergent majorimmunoreactive glycoprotein orthologs of E. canis (36-kD) and E.chaffeensis (47-kD). These glycoproteins have tandem repeat units thatcomprise major B cell epitopes with carbohydrate determinants, whichcontribute substantially to the immunoreactivity of these proteins.Differential expression of these glycoproteins was observed only ondense-cored morphological forms of the bacterium, and the gp36 and gp47are surface-exposed and secreted extracellularly, in certain aspects ofthe invention.

Specifically, a polynucleotide encoding a major immunoreactive 36 kDaprotein of E. canis was identified by a genomic library screen.Recombinant protein reacted strongly with immune dog sera, migratedlarger than predicted by SDS-PAGE, and carbohydrate was detected,demonstrating that the protein was glycosylated. The E. chaffeensis gp47ortholog was discovered by BLAST searching its genome sequence, andrecombinant protein exhibited similar characteristics. Immunoelectronmicroscopy determined that E. canis gp36 and E. chaffeensis gp47 wereexpressed on the surface of the infectious dense-cored forms of thebacteria, but not the metabolically-active reticulate forms. Thepolynucleotide encoding E. canis gp36 contains six tandem repeatsencoding nine amino acids, and at least some of the serines andthreonines in the tandem repeats are glycosylation sites, in specificembodiments of the invention. A single repeat unit expressed as a fusionprotein was sufficient for glycosylation and recognition by immune dogserum. In specific embodiments of this invention, these modificationslend support to protein immunogenicity and function. By ELISA, forexample, synthetic peptide of the 9-mer repeat withoutpost-translational modification was not recognized by antiserum againstE. canis, and periodate treatment of the fusion protein to modifycarbohydrate structures significantly reduced the antibody binding.

Thus, in embodiments of the invention novel major immunoreactive proteinorthologs from E. canis and E. chaffeensis were identified that aredifferentially expressed glycoproteins with tandem repeats. The nineamino acid E. canis gp36 repeat region is an antibody epitope thatrequires carbohydrate for recognition, in particular aspects of theinvention. The present invention provides the first demonstration ofdependence on glycosylation for antibody recognition of an ehrlichialprotein and that the E. coli can modify proteins in a similar manner toEhrlichia.

Furthermore, acidic residues in the repeat region also affectedmobility, a consensus that, in specific aspects of the invention,concerns these proteins as being modified on “Yin-Yang” sites withphosphorylation in addition to glycosylation. The mucin-like protein ofE. ruminantium was recently described to act as an adhesin for tickcells. In specific embodiments, the post-translational modificationscontribute to protein immunogenicity as well as adhesin function, makingmucin-like proteins useful for ehrlichial subunit vaccines. Periodatetreatment of the fusion protein to modify carbohydrate structuresreduced the antibody binding, demonstrating partial dependence onglycosylation for recognition.

In specific aspects of the present invention, there are ehrlichialpolypeptide compositions (or polynucleotide compositions that encode allor part of them) with one or more of the following characteristics: 1)comprises one or more carbohydrate moities, which in specificembodiments comprises part of an epitope determinant; 2) comprises aboutfour to about sixteen tandem repeats, although fewer or more than thisrange may be present; 3) comprises one or more moieties, such as anepitope, that are immunogenically species-specific; 4) is releasedextracellularly, such as by secretion; 5) comprises major B cellepitopes; 6) is surface-exposed; 7) is associated with the infectiousdense-cored forms of ehrlichiae, such as on the surface, for example; 8)is associated with morula membranes (ehrlichiae organisms formmicrocolonies inside cellular vacuoles (morulae) that harbor manyindividual ehrlichiae) comprising dense-cored forms; 9) comprisesvirulence factor activity; and 10) comprises adhesin activity. Infurther aspects, recombinant polypeptide compositions of the presentinvention are able to be glycosylated in a cell to which it is notnative, such as an E. coli cell, for example. The recombinantpolypeptide may then be employed as an immunogenic composition,including, for example, a vaccine.

In particular embodiments of the invention, there are E. canisimmunogenic compositions that comprise an amino acid sequence that isimmunogenic, and in further particular embodiments, the immunogenicityis characterized by being at least part of an epitope. In furtherembodiments, the amino acid sequence comprises at least part of avaccine composition against an ehrlichial organism, such as E. canis. Inspecific embodiments, the amino acid sequence comprises serines,threonines, or, optionally, alanine, proline, valine, and/or glutamicacid; in additional embodiments, the amino acid sequence isglycosylated. In further specific embodiments, the amino acid sequencecomprises part or all of the following exemplary sequence: TEDSVSAPA(SEQ ID NO:22). In other embodiments, the E. canis immunogeniccomposition comprises part or all of the exemplary SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, or mixtures thereof. In other embodiments, the E.canis immunogenic composition is encoded by part or all of SEQ ID NO:32,SEQ ID NO:33, or SEQ ID NO:34, for example. In additional embodiments,the amino acid sequence is comprised in a pharmaceutically acceptableexcipient, which in some aspects of the invention comprises an adjuvant.

In particular embodiments of the invention, there are E. chaffeensisimmunogenic compositions that comprise an amino acid sequence that isimmunogenic, and in further particular embodiments, the immunogenicityis characterized by being at least part of an epitope. In furtherembodiments, the amino acid sequence comprises at least part of avaccine against an ehrlichial organism, such as E. chaffeensis. Inspecific embodiments, the amino acid sequence comprises serines,threonines, or, optionally, alanine, proline, valine, and/or glutamicacid; in additional embodiments the amino acid sequence is glycosylated.In further specific embodiments, the amino acid sequence comprises partor all of the following sequences or a mixture thereof:ASVSEGDAVVNAVSQETPA (SEQ ID NO:23); andEGNASEPVVSQEAAPVSESGDAANPVSSSENAS (SEQ ID NO:24); these exemplarysequences were identified in different E. chaffeensis strains. Other E.chaffeensis sequences may be identified following sequencing of gp47 inother strains; additional E. chaffeensis strains are tested, including91HE17, Jax, St. Vincent, Sapulpa, and V1-V8, for example. In otherembodiments, the E. chaffeensis immunogenic composition comprises partor all of SEQ ID NO:40, SEQ ID NO:41, or mixtures thereof. In additionalembodiments, the E. chaffeensis immunogenic compositions are encoded bypart or all of SEQ ID NO:35 or SEQ ID NO:36, for example. In additionalembodiments, the amino acid sequence is comprised in a pharmaceuticallyacceptable excipient, which in some aspects of the invention comprisesan adjuvant.

In certain embodiments of the present invention, there are immunogenicgp36 E. canis compositions, and particular sequences of the gp36compositions may impart its immunogenicity; for example, a region of thegp36 composition may comprise an epitope. In particular embodiments, oneor more epitopes on a gp36 composition are located in the C-terminus ofa gp36 polypeptide. In some aspects of the invention, multiple differentE. canis strains comprise immunogenic gp36 compositions, and there issignificant sequence identity among the strains in regions of the gp36compositions that comprise the epitope (such as greater than about 80%,85%, 90%, 95%, or 98%, for example). However, in some embodiments, theremay be significant sequence identity among the strains in regions of thegp36 compositions that do not comprise the epitope. In particularaspects of the invention, there is a gp36 composition that isimmunogenic for more than one strain of E. canis, including, forexample, North Carolina (Jake), Oklahoma, and North Carolina (Demon.)Other E. canis strains that may comprise a gp36 immunogenic compositioninclude North Carolina (DJ), North Carolina (Fuzzy), Louisiana, Fla.,and in particular aspects the epitope of the other strains is SEQ IDNO:22, although other epitopes may also be identified. In embodimentswherein an alternative gp36 E. canis epitope to SEQ ID NO:22 isidentified, there may be provided an immunogenic composition comprisinga mixture of gp36 E. canis epitopes, such as a mixture including SEQ IDNO:22, for example.

In certain embodiments of the present invention, there are immunogenicgp47 E. chaffeensis compositions, and particular sequences of the gp47compositions may impart its immunogenicity; for example, a region of thegp47 composition may comprise an epitope. In particular embodiments, oneor more epitopes on a gp47 composition are located in the C-terminus ofa gp47 polypeptide. In some aspects of the invention, multiple differentE. chaffeensis strains comprise immunogenic gp47 compositions, and thereis significant sequence identity among the strains in regions of thegp47 compositions that comprise the epitope (such as greater than about80%, 85%, 90%, 95%, or 98%, for example). However, in some embodiments,there may be significant sequence identity among the strains in regionsof the gp47 compositions that do not comprise the epitope. In additionalembodiments, there may not be significant sequence identity among thedifferent strains in regions of the gp47 compositions that comprise anepitope. Thus, there may be provided an immunogenic compositioncomprising a mixture of gp47 E. chaffeensis epitopes, such as a mixtureincluding SEQ ID NO:23 and/or SEQ ID NO:24, for example. However, inparticular aspects of the invention, there is a gp47 composition that isimmunogenic for more than one strain of E. chaffeensis.

In certain embodiments of the invention, immunogenic compositions of E.canis and E. chaffeensis comprise one or more carbohydrate moities. Inparticular aspects, the carbohydrate moieties facilitate the immunogenicnature of the composition. In specific embodiments, the carbohydratemoiety is required for immunogenicity, whereas in alternativeembodiments the carbohydrate moiety enhances immunogenicity. Thecarbohydrate moiety may be of any kind, so long as it is suitable toallow or enhance immunogenicity. The identity of a carbohydrate moietymay be determined by any suitable means in the art, although inparticular aspects an enzyme that cleaves particular carbohydrates frompolypeptides or peptides, followed by analysis of the cleavedcarbohydrate, for example with mass spectroscopy, may be utilized. Inother means, the carbohydrate is removed and assayed with a variety oflectins, which are known to bind specific sugars.

In specific embodiments of the invention, one or more carbohydratemoieties on the glycoprotein are identified by suitable method(s) in theart, for example gas chromatography/mass spectrometry.

In an embodiment of the invention, there is an immunogenic gp36 E. canisglycoprotein. In another embodiment of the invention, there is animmunogenic gp47 E. chaffeensis glycoprotein. In an additionalembodiment of the invention, there is an E. canis composition comprisingSEQ ID NO:22. In specific aspects of the invention, the compositionfurther comprises a pharmaceutically acceptable excipient. Thecomposition may be further defined as comprising one or morecarbohydrate moieties, as comprising part or all of an epitope, and/oras a vaccine, such as a subunit vaccine.

In an additional embodiment of the invention, there is an E. chaffeensiscomposition comprising SEQ ID NO:23. In a specific embodiment, thecomposition further comprises a pharmaceutically acceptable excipient.The composition may be further defined as comprising one or morecarbohydrate moieties, as comprising part or all of an epitope, and/oras a vaccine, such as a subunit vaccine.

In another embodiment of the invention, there is an E. chaffeensiscomposition comprising SEQ ID NO:24. In a specific embodiment, thecomposition further comprises a pharmaceutically acceptable excipient.The composition may be further defined as comprising one or morecarbohydrate moieties, as comprising part or all of an epitope, and/oras a vaccine, such as a subunit vaccine.

In another embodiment of the invention, there is an E. canis compositioncomprising a polypeptide encoded by at least part of the polynucleotideof SEQ ID NO:32; an E. canis composition comprising a polypeptideencoded by at least part of the polynucleotide of SEQ ID NO:33; an E.canis composition comprising a polypeptide encoded by at least part ofthe polynucleotide of SEQ ID NO:34; an E. chaffeensis compositioncomprising a polypeptide encoded by at least part of the polynucleotideof SEQ ID NO:35; or an E. chaffeensis composition comprising apolypeptide encoded by at least part of the polynucleotide of SEQ IDNO:36.

In a specific embodiment, there is an isolated polynucleotide thatencodes SEQ ID NO:22, an isolated polynucleotide that encodes SEQ IDNO:23, and isolated polynucleotide that encodes SEQ ID NO:24, anisolated polynucleotide of SEQ ID NO:32, an isolated polynucleotide ofSEQ ID NO:33, an isolated polynucleotide of SEQ ID NO:34, an isolatedpolynucleotide of SEQ ID NO:35, and/or an isolated polynucleotide of SEQID NO:36.

In particular embodiments, there is an isolated polynucleotide,comprising: a) a polynucleotide that encodes SEQ ID NO:37; or b) apolynucleotide that is at least about 90% identical to thepolynucleotide of a) and that encodes an immunoreactive E. canis gp36polypeptide. In a specific embodiment, the polynucleotide is furtherdefined as SEQ ID NO:32.

In a further embodiment of the invention, there is an isolatedpolynucleotide, comprising: a) a polynucleotide that encodes SEQ IDNO:38; or b) a polynucleotide that is at least about 90% identical tothe polynucleotide of a) and that encodes an immunoreactive E. canisgp36 polypeptide. In a specific embodiment, the polynucleotide isfurther defined as SEQ ID NO:33. In additional aspects of the invention,there is an isolated polynucleotide, comprising: a) a polynucleotidethat encodes SEQ ID NO:39; or b) a polynucleotide that is at least about90% identical to the polynucleotide of a) and that encodes animmunoreactive E. canis gp36 polypeptide. In a specific embodiment thepolynucleotide is further defined as SEQ ID NO:34.

In an additional particular embodiment, there is an isolatedpolynucleotide, comprising: a) a polynucleotide that encodes SEQ IDNO:40; or b) a polynucleotide that is at least about 90% identical tothe polynucleotide of a) and that encodes an immunoreactive E.chaffeensis gp47 polypeptide. In a specific embodiment, thepolynucleotide is further defined as SEQ ID NO:35.

In another particular embodiment, there is an isolated polynucleotide,comprising: a) a polynucleotide that encodes SEQ ID NO:41; or b) apolynucleotide that is at least about 90% identical to thepolynucleotide of a) and that encodes an immunoreactive E. chaffeensisgp47 polypeptide. In a specific embodiment, the polynucleotide isfurther defined as SEQ ID NO:36.

Polynucleotides of the invention may be comprised in a vector, such as aviral vector or a non-viral vector. An exemplary viral vector includesan adenoviral vector, a retroviral vector, a lentiviral vector, anadeno-associated vector, a herpes virus vector, or a vaccinia virusvector. In a specific embodiment, the non-viral vector is a plasmid.Polynucleotides may be comprised in and/or with liposomes. In a specificaspect, vectors comprise a promoter operably linked to thepolynucleotide, such as one operable in a prokaryote, a eukaryote, orboth, for example. Polynucleotides may be comprised in apharmaceutically acceptable excipient.

In an additional embodiment of the invention, there is an isolatedpolypeptide, comprising: a) SEQ ID NO:22; or b) a gp36 polypeptide thatis at least about 70% identical to SEQ ID NO:22 and that comprisesimmunogenic activity. In a specific embodiment, the polypeptide iscomprised in a pharmaceutically acceptable excipient, and/or it may befurther defined as being comprised in a pharmaceutical compositionsuitable as a vaccine.

In another aspect of the invention, there are isolated antibodies thatbind one or more polypeptides of the invention. Antibodies may bemonoclonal, polyclonal, or antibody fragments, for example.

In an additional embodiment of the invention, there is an isolatedpolypeptide, comprising: a) SEQ ID NO:23 or SEQ ID NO:24; or b) a gp47polypeptide that is at least about 70% identical to SEQ ID NO:23 or SEQID NO:24 and that comprises immunogenic activity. The polypeptide may becomprised in a pharmaceutically acceptable excipient and/or may befurther defined as being comprised in a pharmaceutical compositionsuitable as a vaccine.

In an additional embodiment of the invention, there is a method ofproviding resistance to E. canis or E. chaffeensis infection in anindividual, comprising the step of delivering a therapeuticallyeffective amount of a respective gp36 or gp47 antibody of the inventionto the individual.

In another embodiment, there is a method of inducing an immune responsein an individual, comprising the step of delivering to the individual atherapeutically effective amount of a gp36 or gp47 polypeptide of of theinvention. In an additional embodiment of the present invention, thereis a method of inhibiting or preventing E. canis infection in a subjectcomprising the steps of: identifying a subject prior to exposure orsuspected of being exposed to or infected with E. canis; andadministering a polypeptide of the invention in an amount effective toinhibit E. canis infection.

In one aspect of the present invention, there is a method of inhibitingor preventing E. chaffensis infection in a subject comprising the stepsof: identifying a subject prior to exposure or suspected of beingexposed to or infected with E. chaffeensis; and administering apolypeptide of the invention in an amount effective to inhibit E.chaffeensis infection. In other aspects of the invention, there is amethod of identifying an E. canis infection in an individual, comprisingthe steps of: providing a sample from the individual; and assaying thesample for an antibody of the invention.

In an additional aspect of the invention, there is a method ofidentifying an E. chaffeensis infection in an individual, comprising thesteps of: providing a sample from the individual; and assaying thesample for an antibody of the invention.

In one embodiment of the invention, there is an isolated compositioncomprising an Ehrlichia gp36 or gp47 glycoprotein, comprising: (a) asequence selected from the group consisting of SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:53; SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, and SEQ ID NO:61; (b) a sequence selected from the groupconsisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:63, SEQ ID NO:65,SEQ ID NO:67, and SEQ ID NO:69; or (c) a sequence that is at least about70% identical to one or more sequences in (a) or (b). The compositionmay be further defined as a sequence that is at least about 75%, about80%, about 85%, about 90%, or about 95% identical to one or moresequences in (a) or (b).

The composition may be further defined as comprising a sequence selectedfrom the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:53; SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:61and, optionally, it further comprises SEQ ID NO:22. The composition mayalso be further defined as comprising a sequence selected from the groupconsisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:63, SEQ ID NO:65,SEQ ID NO:67, and SEQ ID NO:69 and, optionally, it further comprises oneor both of SEQ ID NO:23 and SEQ ID NO:24. The composition may also befurther defined as being comprised in a pharmaceutically acceptableexcipient, as comprising two or more carbohydrate moieties, and/or asbeing comprised in a pharmaceutical composition suitable as a vaccine.

In some aspects of the invention the composition may be encoded by apolynucleotide comprising: (a) a polynucleotide selected from the groupconsisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and SEQ ID NO:60; (b) apolynucleotide selected from the group consisting of SEQ ID NO:35, SEQID NO:36, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:68;(c) a polynucleotide that is at least about 70% identical to apolynucleotide of (a) or (b) and encodes an immunoreactive E. canis gp36or gp47 polypeptide; or (d) a polynucleotide that hybridizes to one ormore polynucleotides of (a), (b), or (c) under stringent conditions. Inspecific embodiments of the invention, the polynucleotide of (c) is atleast about 70% identical, at least about 75% identical, at least about80% identical, at least about 85% identical, at least about 90%identical, or at least about 95% identical to a polynucleotide of (a) or(b) and encodes an immunoreactive E. canis gp36 or gp47 polypeptide.

In another embodiment of the invention, there is an isolated Ehrlichiapolynucleotide, comprising: a) a polynucleotide selected from the groupconsisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, and SEQ ID NO:60; (b) apolynucleotide selected from the group consisting of SEQ ID NO:35, SEQID NO:36, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, and SEQ ID NO:68;(c) a polynucleotide that is at least about 70% identical, at leastabout 75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, or at least about 95% identicalto a polynucleotide of (a) or (b) and encodes an immunoreactive E. canisgp36 or gp47 polypeptide; or (d) a polynucleotide that hybridizes to oneor more polynucleotides of (a), (b), or (c) under stringent conditions.

The polynucleotide may be comprised in a vector, such as a viral vectoror a non-viral vector, wherein the viral vector may be an adenoviralvector, a retroviral vector, a lentiviral vector, an adeno-associatedvector, a herpes virus vector, or a vaccinia virus vector and whereinthe non-viral vector may be a plasmid. In further aspects of theinvention, the vector comprise a promoter operably linked to thepolynucleotide wherein the promoter is operable in a prokaryote, aeukaryote, or both. The polynucleotide of the invention may be comprisedin a liposome and/or comprised in a pharmaceutically acceptableexcipient.

In certain aspects of the invention, there is an isolated antibody thatreacts immunologically to a polypeptide of the invention, and theantibody may be a monoclonal antibody, may be comprised in polyclonalantisera, or may be an antibody fragment, for example.

In other embodiments of the invention, there is a method of inducing animmune response in an individual, comprising the step of delivering tothe individual a therapeutically effective amount of a polypeptide ofthe invention.

In additional embodiments of the invention, there is a method ofinhibiting E. canis or E. chaffeensis infection in a subject comprisingthe steps of: identifying a subject prior to exposure or suspected ofbeing exposed to or infected with E. canis or E. chaffeensis; andadministering the polypeptide of the invention in an amount effective toinhibit E. canis or E. chaffeensis infection. In further embodiments ofthe invention, there is a method of identifying an E. canis or E.chaffeensis infection in an individual, comprising the steps of:providing a sample from the individual; and assaying the sample for anantibody of the invention.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features that are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 illustrates genetic organization of the known “mucin-like”orthologs of E. canis and E. chaffeensis. White bars represent thetandem repeat regions and the gray bars represent length (base pairs) ofregions upstream or downstream of the tandem repeats. E. canis strainsillustrated include Jake (Ja), Oklahoma (Ok), and Demon (Dem), and E.chaffeensis strains include Arkansas (Ark) and Sapulpa (Sap).

FIGS. 2A-2B provide immunoreactivity and carbohydrate detection ofrecombinant gp36 and gp47. In FIG. 2A, western immunoblot of recombinantgp36 reacted with anti-E. canis dog serum (lane 1), and carbohydratedetection (lane 2). In FIG. 2B, western immunoblot of recombinant gp47reacted with anti-E. chaffeensis dog serum (lane 1) and carbohydratedetection (lane 2).

FIGS. 3A-3B show immunoblots of E. canis or E. chaffeensis lysate withdifferent compositions. In FIG. 3A, there is western immunoblot of E.canis lysate with gp36 with anti-recombinant gp36 (lane 1) and anti-E.canis dog serum (lane 2). In FIG. 3B, there is reactivity of E.chaffeensis lysate with HME patient sera (lanes 1-10), anti-recombinantE. chaffeensis gp47, and anti-E. chaffeensis dog serum.

FIG. 4. shows kinetic antibody responses to E. canis gp36 (days 0, 14and 56) of 15 dogs (lanes 1-15) experimentally infected with E. canis.

FIGS. 5A-5C provide western immunoblot of thioredoxin control (FIGS. 5A,5B, 5C, lane 1) and the E. canis gp36 single repeat fusion protein (9amino acids) (FIGS. 5A, 5B, lane 2) probed with anti-thioredoxin (PanelA) and anti-E. canis dog serum (FIG. 5B). Western immunoblot of the E.chaffeensis gp47 single repeat fusion protein (19 amino acids) (FIG. 5C,lane 2) probed with anti-E. chaffeensis dog serum (FIG. 5C).

FIGS. 6A-6B shows that western immunoblot of E. canis gp36 and E.chaffeensis gp47 reacted with homologous and heterologous antibody.Native E. canis gp36 (lane 1), gp36 single repeat recombinant protein(lane 2), native E. chaffeensis gp47 (lane 3) and gp47 single repeatrecombinant protein reacted with anti-recombiant gp36 (FIG. 6A) andanti-recombinant gp47 (FIG. 6B) antibody.

FIGS. 7A-7C show contribution of glycans to the antibody reactivity ofE. canis Jake strain gp36 and E. chaffeensis gp47 as determined byELISA. FIG. 7A shows antibody reactivities of untreated andperiodate-treated recombinant E. canis gp36 with anti-E. canis dog serum(#2995). FIG. 7B shows immunoreactivities of the recombinant E. canisgp36 repeat fusion peptides containing the 9-mer, 12-mer, and 18-mercompared to those of aglycosylated synthetic peptides. FIG. 7C showsimmunoreactivities of the recombinant E. chaffeensis gp47 repeat fusionpeptide (19-mer) and aglycosylated synthetic peptide with anti-E.chaffeensis dog serum (#2495). OD, optical density

FIGS. 8A-8B show an immunogold-labeled electronmicrograph of E. canisgp36 and E. chaffeensis gp47. In FIG. 8A, there is localization of gp36in morulae containing the E. canis reticulate (R) or the dense-cored(DC) morphological forms. In FIG. 8B, there is localization of gp47 inmorulae containing the E. chaffeensis reticulate (R) or the dense-cored(DC) morphological forms.

FIGS. 9A-9B show secreted E. canis and E. chaffeensis immunoreactiveproteins. In FIG. 9A, there is western immunoblot of concentratedsupernatants from E. canis infected DH82 with anti-E. canis dog serum(lane 1) and anti-recombinant E. canis gp36 (lane 2). In FIG. 9B, thereis western immunoblot of supernatants from E. chaffeensis-infected DH82cells with anti-E. chaffeensis dog serum (lane 1) and withanti-recombinant E. chaffeensis gp47 (lane 2).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The term “adhesin” as used herein refers to a composition that mediatesadhesion of a bacterium to a surface, such as to play a role inpathogenesis. In specific aspects of the invention, adhesins arebacterial surface antigens that often exist in the form of filamentousprojections (pili or fimbriae, for example) and bind to specific cellreceptors, such as those on epithelial cell membranes.

The term “carbohydrate” as used herein refers to a composition comprisedof carbon, hydrogen, and oxygen, particularly in the ratio of 2H:1C:1O.The term includes sugars, starches, and celluloses, for example.

The term “epitope” as used herein refers to a site of a composition towhich a specific antibody binds.

The term “glycan,” which may also be referred to as a “polysaccharide,”as used herein refers to a carbohydrate that can be decomposed byhydrolysis into two or more monosaccharides. In other words, it may bereferred to as a chain of simple sugars (aldehyde or ketone derivativesof a polyhydric alcohol).

The term “immunogenic” as used herein refers to a composition that isable to provoke an immune response against it.

The term “immune response” as used herein refers to the reaction of theimmune system to the presence of an antigen by making antibodies to theantigen. In further specific embodiments, immunity to the antigen may bedeveloped on a cellular level, by the body as a whole, hypersensitivityto the antigen may be developed, and/or tolerance may be developed, suchas from subsequent challenge. In specific embodiments, an immuneresponse entails lymphocytes identifying an antigenic molecule asforeign and inducing the formation of antibodies and lymphocytes capableof reacting with it and rendering it less harmful.

The term “immunoreactive” as used herein refers to a composition beingreactive with antibodies from the sera of an individual. In specificembodiments, a composition is immunoreactive if an antibody recognizesit, such as by binding to it.

The term “mucin” as used herein refers to one or more highlyglycosylated glycoproteins with N-acetylgalactosamine (GalNAc.)

The term “ortholog” as used herein refers to a polynucleotide from onespecies that corresponds to a polynucleotide in another species; the twopolynucleotides are related through a common ancestral species (ahomologous polynucleotide). However, the polynucleotide from one specieshas evolved to become different from the polynucleotide of the otherspecies.

The term “subunit vaccine” as used herein refers to a vaccine wherein apolypeptide or fragment thereof is employed, as opposed to an entireorganism.

The term “vaccine” as used herein refers to a composition that providesimmunity to an individual upon challenge.

The term “virulence factor” as used herein refers to one or more geneproducts that enable a microorganism to establish itself on or within aparticular host species and enhance its pathogenicity. Exemplaryvirulence factors include, for example, cell surface proteins thatmediate bacterial attachment, cell surface carbohydrates and proteinsthat protect a bacterium, bacterial toxins, and hydrolytic enzymes thatmay contribute to the pathogenicity of the bacterium.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and so forth which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL,Second Edition (1989), OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait Ed., 1984),ANIMAL CELL CULTURE (R. I. Freshney, Ed., 1987), the series METHODS INENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIANCELLS (J. M. Miller and M. P. Calos eds. 1987), HANDBOOK OF EXPERIMENTALIMMUNOLOGY, (D. M. Weir and C. C. Blackwell, Eds.), CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore,J. G. Siedman, J. A. Smith, and K. Struhl, eds., 1987), CURRENTPROTOCOLS IN IMMUNOLOGY (J. E. coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach and W. Strober, eds., 1991); ANNUAL REVIEW OFIMMUNOLOGY; as well as monographs in journals such as ADVANCES INIMMUNOLOGY. All patents, patent applications, and publications mentionedherein, both supra and infra, are hereby incorporated herein byreference.

II. The Present Invention

The present invention concerns compositions and methods related toEhrlichia spp. proteins and the polynucleotides that encode them. Inparticular aspects of the invention, there are differentially-expressedand secreted major immunoreactive protein orthologs of E. canis and E.chaffeensis that elicit early antibody responses to epitopes onglycosylated tandem repeats. Specifically, the present inventionconcerns one or more glycoproteins from Ehrlichia spp., in specificembodiments. In further embodiments, the present invention relates to aglycoprotein from Ehrlichia spp. that is a gp36 protein. In additionalembodiments, the gp36 protein is from E. canis. In additionalembodiments, the present invention relates to a glycoprotein fromEhrlichia spp. that is a gp47 protein. In additional embodiments, thegp47 protein is from E. chaffeensis.

In specific aspects of the invention, two E. canis major immunoreactiveproteins, 36- and 19-kDa, elicit the earliest detectable antibodyresponse during the acute phase of canine monocytic ehrlichiosis. Genesencoding the major immunoreactive 36 kDa protein of E. canis and acorresponding ortholog of E. chaffeensis (47-kDa) were identified, andtheir immunoreactivity and expression were determined. Consistent withother ehrlichial immunoreactive proteins, carbohydrate was detected onthe recombinant gp36 and gp47, and their masses were substantiallylarger than predicted (26.7- and 32.9-kDa, respectively). The E. canisgp36 and E. chaffeensis gp47 each have carboxy-terminal tandem repeatunits (about four to sixteen repeats) that varied in number and aminoacid sequences among different isolates. Species-specific antibodyepitopes were identified in the C-terminal non-homologous repeatcomprising regions of gp36 and gp47, and recombinant single repeat unitsfrom both were recognized by antibody. However, a homologous syntheticpeptide repeat unit from E. canis gp36 was not immunoreactive, andperiodate treatment of the immunoreactive recombinant peptidesubstantially reduced the antibody reactivity, demonstrating thatglycans are important epitope determinants that are structurallyconserved on the recombinant proteins expressed in E. coli. The E. canisgp36 and E. chaffeensis gp47 were differentially expressed only on thesurface of dense-cored ehrlichiae. Furthermore, gp36 and gp47 weredetected in the ehrlichia-free supernatants, indicating that theseproteins are released extracellularly during infection. This inventionconcerns these newly identified glycoproteins as immunogeniccompositions, such as vaccines, including subunit vaccines, orimmunodiagnostic antigens, for example.

Some embodiments of the present invention are directed toward a methodof inhibiting E. canis infection in a subject comprising the steps ofidentifying a subject prior to exposure or suspected of being exposed toor infected with E. canis; and administering a composition comprising a36-kDa antigen of E. canis in an amount effective to inhibit E. canisinfection. The inhibition may occur through any means such as e.g., thestimulation of the subject's humoral or cellular immune responses, or byother means such as inhibiting the normal function of the 36-kDaantigen, or even competing with the antigen for interaction with someagent in the subject's body, or a combination thereof, for example.

Some embodiments of the present invention are directed toward a methodof inhibiting E. chaffeensis infection in a subject comprising the stepsof identifying a subject prior to exposure or suspected of being exposedto or infected with E. chaffeensis; and administering a compositioncomprising a 47-kDa antigen of E. chaffeensis in an amount effective toinhibit E. chaffeensis infection. The inhibition may occur through anymeans such as e.g., the stimulation of the subject's humoral or cellularimmune responses, or by other means such as inhibiting the normalfunction of the 47-kDa antigen, or even competing with the antigen forinteraction with some agent in the subject's body, or a combinationthereof, for example.

The present invention is also directed toward a method of targetedtherapy to an individual, comprising the step of administering acompound to an individual, wherein the compound has a targeting moietyand a therapeutic moiety, and wherein the targeting moiety is specificfor gp36 protein. In certain aspects, the targeting moiety is anantibody specific for gp36 or ligand or ligand binding domain that bindsgp36. Likewise, the therapeutic moiety may comprise a radioisotope, atoxin, a chemotherapeutic agent, an immune stimulant, a cytotoxic agent,or an antibiotic, for example.

The present invention is also directed toward a method of targetedtherapy to an individual, comprising the step of administering acompound to an individual, wherein the compound has a targeting moietyand a therapeutic moiety, and wherein the targeting moiety is specificfor gp47 protein. In certain aspects, the targeting moiety is anantibody specific for gp47 or ligand or ligand binding domain that bindsgp47. Likewise, the therapeutic moiety may comprise a radioisotope, atoxin, a chemotherapeutic agent, an immune stimulant, a cytotoxic agent,or an antibiotic, for example.

Other embodiments of the present invention concern diagnosis ofehrlichial infection in a mammal by assaying a sample from the mammal,such as blood or serum, for example, for antibodies to a gp36composition (for E. canis) or to a gp47 composition (for E.chaffeensis).

III. E. canis gp36 and E. chaffeensis gp47 Amino Acid Compositions

The present invention regards a polypeptide comprising E. canis gp36, E.chaffeensis gp47, or a mixture thereof. For the sake of brevity, thefollowing section will refer to any E. canis gp36 and/or E. chaffeensisgp47 amino acid compositions of the present invention, includingpolypeptides and peptides.

In particular embodiments, there is an amino acid sequence, wherein thepolypeptide may be a recombinant protein or it may be isolated and/orpurified from nature, for example. In particular aspects, the amino acidsequence is encoded by a nucleic acid sequence. The polypeptide isuseful as an antigen, in specific embodiments.

The present invention is also directed towards a method of producing therecombinant protein, comprising the steps of obtaining a vector thatcomprises an expression construct comprising a sequence encoding theamino acid sequence operatively linked to a promoter; transfecting thevector into a cell; and culturing the cell under conditions effective ofthe expression construct. The amino acid sequence may be generatedsynthetically, in alternative embodiments.

By a “substantially pure protein” is meant a protein that has beenseparated from at least some of those components that naturallyaccompany it. A substantially pure immunoreactive composition may beobtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding an immunoreactivecomposition; or by chemically synthesizing the protein, for example.Accordingly, substantially pure proteins include prokaryotic proteinssynthesized in E. coli, other prokaryotes, or any other organism inwhich they do not naturally occur.

Thus, in certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule. As usedherein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 200 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, about60, about 61, about 62, about 63, about 64, about 65, about 66, about67, about 68, about 69, about 70, about 71, about 72, about 73, about74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about88, about 89, about 90, about 91, about 92, about 93, about 94, about95, about 96, about 97, about 98, about 99, about 100, about 110, about120, about 130, about 140, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 275, about 300, about 325, about 350, about 375, about 400,about 425, about 450, about 475, about 500, about 525, about 550, about575, about 600, about 625, about 650, about 675, about 700, about 725,about 750, about 775, about 800, about 825, about 850, about 875, about900, about 925, about 950, about 975, about 1000, about 1100, about1200, about 1300, about 1400, about 1500, about 1750, about 2000, about2250, about 2500 or greater amino acid residues, and any range derivabletherein.

As used herein, an “amino acid molecule” refers to any polypeptide,polypeptide derivitive, or polypeptide mimetic as would be known to oneof ordinary skill in the art. In certain embodiments, the residues ofthe proteinaceous molecule are sequential, without any non-amino acidmolecule interrupting the sequence of amino acid molecule residues. Inother embodiments, the sequence may comprise one or more non-aminomolecule moieties. In particular embodiments, the sequence of residuesof the proteinaceous molecule may be interrupted by one or morenon-amino molecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3-Aminoadipicacid Hyl Hydroxylysine Bala β-alanine, β-Amino- AHyl allo-Hydroxylysinepropionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIleallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

In certain embodiments the proteinaceous composition comprises at leastone protein, polypeptide or peptide. In further embodiments, theproteinaceous composition comprises a biocompatible protein, polypeptideor peptide. As used herein, the term “biocompatible” refers to asubstance that produces no significant untoward effects when applied to,or administered to, a given organism according to the methods andamounts described herein. Such untoward or undesirable effects are thosesuch as significant toxicity or adverse immunological reactions.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials, for example. Thenucleotide and protein, polypeptide and peptide sequences for variousgenes have been previously disclosed, and may be found at computerizeddatabases known to those of ordinary skill in the art. Two suchdatabases are the National Center for Biotechnology Information'sGenbank and GenPept databases, for example. The coding regions for theseknown genes may be amplified and/or expressed using the techniquesdisclosed herein or as would be know to those of ordinary skill in theart. Alternatively, various commercial preparations of proteins,polypeptides and peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified.Generally, “purified” will refer to a specific or protein, polypeptide,or peptide composition that has been subjected to fractionation toremove various other proteins, polypeptides, or peptides, and whichcomposition substantially retains its activity, as may be assessed, forexample, by the protein assays, as would be known to one of ordinaryskill in the art for the specific or desired protein, polypeptide orpeptide. Exemplary activities that may be assessed for retention in thepurified proteinaceous composition are iron-binding activity andimmunoreactivity.

In specific embodiments of the present invention, a polypeptide islabeled, and any detectable label is suitable in the invention. Thelabel may be attached to the polypeptide at the N-terminus, at theC-terminus, or in a side chain of an amino acid residue. One or morelabels may be employed. Exemplary labels included radioactive labels,fluorescent labels, colorimetric labels, and so forth. In specificembodiments, the label is covalently attached to the polypeptide.

IV. E. canis gp36 and E. chaffeensis gp47 Nucleic Acid Compositions

Certain embodiments of the present invention concern an E. canis gp36and/or an E. chaffeensis gp47 nucleic acid. For the sake of brevity, thefollowing section will refer to any E. canis gp36 and/or E. chaffeensisgp47 nucleic acid compositions of the present invention.

In certain aspects, a nucleic acid comprises a wild-type or a mutantnucleic acid. In particular aspects, a nucleic acid encodes for orcomprises a transcribed nucleic acid. In other aspects, a nucleic acidcomprises a nucleic acid segment, or a biologically functionalequivalent thereof. In particular aspects, a nucleic acid encodes aprotein, polypeptide, peptide.

The term “nucleic acid” is well known in the art and may be usedinterchangeably herein with the term “polynucleotide.” A “nucleic acid”as used herein will generally refer to a molecule (i.e., a strand) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 3 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss,” a double stranded nucleic acid by the prefix “ds,”and a triple stranded nucleic acid by the prefix “ts.”

A. Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, carboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moeity. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moieties comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art.

B. Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

C. Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

D. Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. Asused herein a “derivative” refers to a chemically modified or alteredform of a naturally occurring molecule, while the terms “mimic” or“analog” refer to a molecule that may or may not structurally resemble anaturally occurring molecule or moiety, but possesses similar functions.As used herein, a “moiety” generally refers to a smaller chemical ormolecular component of a larger chemical or molecular structure.Nucleobase, nucleoside and nucleotide analogs or derivatives are wellknown in the art, and have been described (see for example, Scheit,1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in U.S. Pat. No. 5,681,947 which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167 which describe nucleic acids incorporating fluorescent analogsof nucleosides found in DNA or RNA, particularly for use as flourescentnucleic acids probes; U.S. Pat. No. 5,614,617 which describesoligonucleotide analogs with substitutions on pyrimidine rings thatpossess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663, 5,872,232and 5,859,221 which describe oligonucleotide analogs with modified5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties) used innucleic acid detection; U.S. Pat. No. 5,446,137 which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165 whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606 which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697 which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847 which describe the linkage of a substituentmoeity which may comprise a drug or label to the 2′ carbon of anoligonucleotide to provide enhanced nuclease stability and ability todeliver drugs or detection moieties; U.S. Pat. No. 5,223,618 whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967 which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240 which describe oligonucleotides with three or four atom linkermoeity replacing phosphodiester backbone moeity used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988 which describes hydrophobic carrier agent attached tothe 2′-O position of oligonuceotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136 which describesolignucleotides conjugaged to anthraquinone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922 which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H; and U.S. Pat. No. 5,708,154 which describes RNA linkedto a DNA to form a DNA-RNA hybrid.

E. Polyether and Peptide Nucleic Acids

In certain embodiments, it is contemplated that a nucleic acidcomprising a derivative or analog of a nucleoside or nucleotide may beused in the methods and compositions of the invention. A non-limitingexample is a “polyether nucleic acid”, described in U.S. Pat. No.5,908,845, incorporated herein by reference. In a polyether nucleicacid, one or more nucleobases are linked to chiral carbon atoms in apolyether backbone.

Another non-limiting example is a “peptide nucleic acid”, also known asa “PNA”, “peptide-based nucleic acid analog” or “PENAM”, described inU.S. Pat. Nos. 5,786,461, 5,891,625, 5,773,571, 5,766,855, 5,736,336,5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which isincorporated herein by reference. Peptide nucleic acids generally haveenhanced sequence specificity, binding properties, and resistance toenzymatic degradation in comparison to molecules such as DNA and RNA(Egholm et al., 1993; PCT/EP/01219). A peptide nucleic acid generallycomprises one or more nucleotides or nucleosides that comprise anucleobase moiety, a nucleobase linker moeity that is not a 5-carbonsugar, and/or a backbone moiety that is not a phosphate backbone moiety.Examples of nucleobase linker moieties described for PNAs include azanitrogen atoms, amido and/or ureido tethers (see for example, U.S. Pat.No. 5,539,082). Examples of backbone moieties described for PNAs includean aminoethylglycine, polyamide, polyethyl, polythioamide,polysulfinamide or polysulfonamide backbone moiety.

In certain embodiments, a nucleic acid analogue such as a peptidenucleic acid may be used to inhibit nucleic acid amplification, such asin PCR, to reduce false positives and discriminate between single basemutants, as described in U.S. Pat. No. 5,891,625. Other modificationsand uses of nucleic acid analogs are known in the art, and areencompassed by the gp36 polynucleotide. In a non-limiting example, U.S.Pat. No. 5,786,461 describes PNAs with amino acid side chains attachedto the PNA backbone to enhance solubility of the molecule. In anotherexample, the cellular uptake property of PNAs is increased by attachmentof a lipophilic group. U.S. application Ser. No. 117,363 describesseveral alkylamino moeities used to enhance cellular uptake of a PNA.Another example is described in U.S. Pat. Nos. 5,766,855, 5,719,262,5,714,331 and 5,736,336, which describe PNAs comprising naturally andnon-naturally occurring nucleobases and alkylamine side chains thatprovide improvements in sequence specificity, solubility and/or bindingaffinity relative to a naturally occurring nucleic acid.

F. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemically synthesis usingphosphotriester, phosphite or phosphoramidite chemistry and solid phasetechniques such as described in EP 266,032, incorporated herein byreference, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 1989,incorporated herein by reference).

G. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al., 1989, incorporatedherein by reference).

In certain aspect, the present invention concerns a nucleic acid that isan isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

H. Nucleic Acid Segments

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are smaller fragments of anucleic acid, such as for non-limiting example, those that encode onlypart of the peptide or polypeptide sequence. Thus, a “nucleic acidsegment” may comprise any part of a gene sequence, of from about 2nucleotides to the full length of the peptide or polypeptide encodingregion.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe generated:

n to n+y

where n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10 mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and soon. For a 15-mer, the nucleic acid segments correspond to bases 1 to 15,2 to 16, 3 to 17 . . . and so on. For a 20-mer, the nucleic segmentscorrespond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on. Incertain embodiments, the nucleic acid segment may be a probe or primer.As used herein, a “probe” generally refers to a nucleic acid used in adetection method or composition. As used herein, a “primer” generallyrefers to a nucleic acid used in an extension or amplification method orcomposition.

I. Nucleic Acid Complements

The present invention also encompasses a nucleic acid that iscomplementary to one or more other nucleic acids. In specificembodiments, for example, a nucleic acid is employed for antisense orsiRNA purposes, such as to inhibit at least partially expression of apolynucleotide.

In particular embodiments the invention encompasses a nucleic acid or anucleic acid segment complementary to the sequence set forth herein, forexample. A nucleic acid is “complement(s)” or is “complementary” toanother nucleic acid when it is capable of base-pairing with anothernucleic acid according to the standard Watson-Crick, Hoogsteen orreverse Hoogsteen binding complementarity rules. As used herein “anothernucleic acid” may refer to a separate molecule or a spatial separatedsequence of the same molecule.

As used herein, the term “complementary” or “complement(s)” also refersto a nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. In certain embodiments, a“complementary” nucleic acid comprises a sequence in which about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, to about 100%, and any rangederivable therein, of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization. In certain embodiments, the term “complementary” refersto a nucleic acid that may hybridize to another nucleic acid strand orduplex in stringent conditions, as would be understood by one ofordinary skill in the art.

In certain embodiments, a “partly complementary” nucleic acid comprisesa sequence that may hybridize in low stringency conditions to a singleor double stranded nucleic acid, or contains a sequence in which lessthan about 70% of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization.

J. Hybridization

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

As used herein “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butprecludes hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mRNA transcript or a nucleic acid segment thereof, and thelike.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl, forexample, at temperatures of about 50° C. to about 70° C. or, forexample, wherein said stringent conditions are hybridization at 50-65°C., 5×SSPC, 50% formamide; wash 50-65° C., 5×SSPC; or wash at 60° C.,0.5×SSC, 0.1% SDS. It is understood that the temperature and ionicstrength of a desired stringency are determined in part by the length ofthe particular nucleic acid(s), the length and nucleobase content of thetarget sequence(s), the charge composition of the nucleic acid(s), andto the presence or concentration of formamide, tetramethylammoniumchloride or other solvent(s) in a hybridization mixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of a nucleic acid towards a targetsequence. In a non-limiting example, identification or isolation of arelated target nucleic acid that does not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

V. Nucleic Acid-Based Expression Systems

In particular embodiments, the present invention concerns apolynucleotide that encodes an immunoreactive Ehrlichiae polypeptide,and also includes delivering the polynucleotide encoding thepolypeptide, or encoded product thereof, to an individual in needthereof, such as an individual infected with Erhlichia and/or anindividual susceptible to being infected with Erhlichia. For the sake ofbrevity, the following section will refer to any E. canis gp36 and/or E.chaffeensis gp47 nucleic acid compositions and/or nucleic acid-basedexpression system of the present invention.

The present invention is directed toward substantially pure and/orisolated DNA sequence encoding an immunoreactive Ehrlichia composition.Generally, the encoded protein comprises an N-terminal sequence, whichmay be cleaved after post-translational modification resulting in theproduction of mature protein.

It is well-known in the art that because of the degeneracy of thegenetic code (i.e., for most amino acids, more than one nucleotidetriplet (codon) codes for a single amino acid), different nucleotidesequences can code for a particular amino acid, or polypeptide. Thus,the polynucleotide sequences of the subject invention include any of theprovided exemplary sequences or a degenerate variant of such a sequence,for example. In particular aspects of the invention, a degeneratevariant comprises a sequence that is not identical to a sequence of theinvention but that still retains one or more properties of a sequence ofthe invention.

As used herein, “substantially pure DNA” means DNA that is not part of amilieu in which the DNA naturally occurs, by virtue of separation(partial or total purification) of some or all of the molecules of thatmilieu, or by virtue of alteration of sequences that flank the claimedDNA. The term therefore includes, for example, a recombinant DNA whichis incorporated into a vector, into an autonomously replicating plasmidor virus, or into the genomic DNA of a prokaryote or eukaryote; or thatexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by polymerase chain reaction (PCR) or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA, which is part of a hybrid gene encoding additionalpolypeptide sequence, e.g., a fusion protein.

The present invention is further directed to an expression vectorcomprising a polynucleotide encoding an immunoreactive Ehrlichiaecomposition and capable of expressing the polynucleotide when the vectoris introduced into a cell. In specific embodiments, the vector comprisesin operable linkage the following: a) an origin of replication; b) apromoter; and c) a DNA sequence coding for the protein.

As used herein “vector” may be defined as a replicable nucleic acidconstruct, e.g., a plasmid or viral nucleic acid. Vectors may be used toamplify and/or express nucleic acid encoding an immunoreactivecomposition of Ehrlichiae. An expression vector is a replicableconstruct in which a nucleic acid sequence encoding a polypeptide isoperably linked to suitable control sequences capable of effectingexpression of the polypeptide in a cell. The need for such controlsequences will vary depending upon the cell selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter and/or enhancer, suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation, for example. Methods that are well-knownto those skilled in the art can be used to construct expression vectorscomprising appropriate transcriptional and translational controlsignals. See for example, the techniques described in Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold SpringHarbor Press, N.Y. A polynucleotide sequence to be expressed and itstranscription control sequences are defined as being “operably linked”if the transcription control sequences effectively control thetranscription of the polynucleotide sequence. Vectors of the inventioninclude, but are not limited to, plasmid vectors and viral vectors.Preferred viral vectors of the invention are those derived fromretroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpesviruses, for example.

In general, expression vectors comprise promoter sequences thatfacilitate the efficient transcription of the polynucleotide to beexpressed, are used in connection with a host cell. As used herein, theterm “host” is meant to include not only prokaryotes but alsoeukaryotes, such as yeast, plant and animal cells. A recombinantpolynucleotide that encodes an immunoreactive composition of Ehrlichiaeof the present invention can be used to transform a host using any ofthe techniques commonly known to those of ordinary skill in the art.Prokaryotic hosts may include E. coli, S. tymphimurium, Serratiamarcescens and Bacillus subtilis. Eukaryotic hosts include yeasts, suchas Pichia pastoris, mammalian cells and insect cells.

The following description concerns exemplary elements, reagents, andmethods for polynucleotides and nucleic acid delivery of an Ehrlichiapolynucleotide.

A. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the betalactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the cell,organelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al., 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

The promoter may be one suitable for use in a prokaryotic cell, aeukaryotic cell, or both. Additionally any promoter/enhancer combination(as per, for example, the Eukaryotic Promoter Data Base EPDB) could alsobe used to drive expression. Use of a T3, T7 or SP6 cytoplasmicexpression system is one possible embodiment.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

9. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™ 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with betagalactosidase, ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

10. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Components of the present invention may comprise aviral vector that encode one or more compositions or other componentssuch as, for example, an immunomodulator or adjuvant. Non-limitingexamples of virus vectors that may be used to deliver a nucleic acid ofthe present invention are described below.

a. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

b. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the compositions of thepresent invention as it has a high frequency of integration and it caninfect nondividing cells, thus making it useful for delivery of genesinto mammalian cells, for example, in tissue culture (Muzyczka, 1992) orin vivo. AAV has a broad host range for infectivity (Tratschin et al.,1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,1988). Details concerning the generation and use of rAAV vectors aredescribed in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference.

c. Retroviral Vectors

Retroviruses have useful as delivery vectors due to their ability tointegrate their genes into the host genome, transferring a large amountof foreign genetic material, infecting a broad spectrum of species andcell types and of being packaged in special cell lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., oneencoding a composition of interest) is inserted into the viral genome inthe place of certain viral sequences to produce a virus that isreplication defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

d. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

e. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

11. Vector Delivery and Cell Transformation

Suitable methods for Ehrlichial nucleic acid delivery for transformationof an organelle, a cell, a tissue or an organism for use with thecurrent invention are believed to include virtually any method by whicha nucleic acid (e.g., DNA) can be introduced into an organelle, a cell,a tissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson etal., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al.,1986; Potter et al., 1984); by calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); byusing DEAE dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau etal., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

a. Ex vivo Transformation

Methods for tranfecting vascular cells and tissues removed from anorganism in an ex vivo setting are known to those of skill in the art.For example, cannine endothelial cells have been genetically altered byretrovial gene tranfer in vitro and transplanted into a canine (Wilsonet al., 1989). In another example, yucatan minipig endothelial cellswere tranfected by retrovirus in vitro and transplated into an arteryusing a double-ballonw catheter (Nabel et al., 1989). Thus, it iscontemplated that cells or tissues may be removed and tranfected ex vivousing the nucleic acids of the present invention. In particular aspects,the transplanted cells or tissues may be placed into an organism. Inpreferred facets, a nucleic acid is expressed in the transplated cellsor tissues.

b. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle,a cell, a tissue or an organism via one or more injections (i.e., aneedle injection), such as, for example, subcutaneously, intradermally,intramuscularly, intervenously, intraperitoneally, etc. Methods ofinjection of vaccines are well known to those of ordinary skill in theart (e.g., injection of a composition comprising a saline solution).Further embodiments of the present invention include the introduction ofa nucleic acid by direct microinjection. Direct microinjection has beenused to introduce nucleic acid constructs into Xenopus oocytes (Harlandand Weintraub, 1985). The amount of composition used may vary upon thenature of the antigen as well as the organelle, cell, tissue or organismused

c. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high voltage electric discharge. In some variantsof this method, certain cell wall degrading enzymes, such as pectindegrading enzymes, are employed to render the target recipient cellsmore susceptible to transformation by electroporation than untreatedcells (U.S. Pat. No. 5,384,253, incorporated herein by reference).Alternatively, recipient cells can be made more susceptible totransformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre B lymphocytes have been transfected with humankappa immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin degrading enzymes (pectolyases) ormechanically wounding in a controlled manner. Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

d. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV 1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

e. DEAE Dextran

In another embodiment, a nucleic acid is delivered into a cell usingDEAE dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

f. Sonication Loading

Additional embodiments of the present invention include the introductionof a nucleic acid by direct sonic loading. LTK fibroblasts have beentransfected with the thymidine kinase gene by sonication loading(Fechheimer et al., 1987).

g. Liposome-Mediated Transfection

In a further embodiment of the invention, an Ehrlichial nucleic acid maybe comprised with a lipid complex such as, for example, comprised in aliposome. Liposomes are vesicular structures characterized by aphospholipid bilayer membrane and an inner aqueous medium. Multilamellarliposomes have multiple lipid layers separated by aqueous medium. Theyform spontaneously when phospholipids are suspended in an excess ofaqueous solution. The lipid components undergo self rearrangement beforethe formation of closed structures and entrap water and dissolvedsolutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL)or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry of liposomeencapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposomemay be complexed or employed in conjunction with nuclear non histonechromosomal proteins (HMG 1) (Kato et al., 1991). In yet furtherembodiments, a liposome may be complexed or employed in conjunction withboth HVJ and HMG 1. In other embodiments, a delivery vehicle maycomprise a ligand and a liposome.

h. Receptor-Mediated Transfection

Still further, a nucleic acid may be delivered to a target cell viareceptor mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

Certain receptor mediated gene targeting vehicles comprise a cellreceptor specific ligand and a nucleic acid binding agent. Otherscomprise a cell receptor specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell specific binding. For example, lactosyl ceramide, a galactoseterminal asialganglioside, have been incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes(Nicolau et al., 1987). It is contemplated that the tissue specifictransforming constructs of the present invention can be specificallydelivered into a target cell in a similar manner.

i. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isincorporated herein by reference). This method depends on the ability toaccelerate DNA coated microprojectiles to a high velocity allowing themto pierce cell membranes and enter cells without killing them (Klein etal., 1987). There are a wide variety of microprojectile bombardmenttechniques known in the art, many of which are applicable to theinvention.

Microprojectile bombardment may be used to transform various cell(s),tissue(s) or organism(s), such as for example any plant species.Examples of species which have been transformed by microprojectilebombardment include monocot species such as maize (PCT Application WO95/06128), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat(U.S. Pat. No. 5,563,055, incorporated herein by reference), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casas et al., 1993; Hagio et al., 1991); as well as a number of dicotsincluding tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783, incorporated herein by reference),sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumesin general (U.S. Pat. No. 5,563,055, incorporated herein by reference).

In this microprojectile bombardment, one or more particles may be coatedwith at least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into a cell(e.g., a plant cell) by acceleration is the Biolistics Particle DeliverySystem, which can be used to propel particles coated with DNA or cellsthrough a screen, such as a stainless steel or Nytex screen, onto afilter surface covered with cells, such as for example, a monocot plantcells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

12. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

A tissue may comprise a host cell or cells to be transformed with acomposition of the invention. The tissue may be part or separated froman organism. In certain embodiments, a tissue may comprise, but is notlimited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood(e.g., lymphocytes), blood vessel, bone, bone marrow, brain, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, liver, lung, lymph node, muscle,neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, smallintestine, spleen, stem cells, stomach, testes, anthers, ascite tissue,cobs, ears, flowers, husks, kernels, leaves, meristematic cells, pollen,root tips, roots, silk, stalks, and all cancers thereof.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokayote (e.g., a eubacteria, an archaea) or aneukaryote, as would be understood by one of ordinary skill in the art(see, for example, webpagehttp://phylogeny.arizona.edu/tree/phylogeny.html).

Numerous cell lines and cultures are available for use as a host cell,and they can be obtained through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials (www.atcc.org). An appropriate host canbe determined by one of skill in the art based on the vector backboneand the desired result. A plasmid or cosmid, for example, can beintroduced into a prokaryote host cell for replication of many vectors.Cell types available for vector replication and/or expressioninclude,but are not limited to, bacteria, such as E. coli (e.g., E. coli strainRR1, E. coli LE392, E. coli B, E. coli×1776 (ATCC No. 31537) as well asE. coli W3110 (F, lambda, prototrophic, ATCC No. 273325), DH5a, JMio9,and KCB, bacilli such as Bacillus subtilis; and other enterobacteriaceaesuch as Salmonella typhimurium, Serratia marcescens, various Pseudomonasspecie, as well as a number of commercially available bacterial hostssuch as SURE® Competent Cells and SOLOPACK Gold Cells (STRATAGENE®, LaJolla). In certain embodiments, bacterial cells such as E. coli LE392are particularly contemplated as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

13. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REXTm(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

It is contemplated that the proteins, polypeptides or peptides producedby the methods of the invention may be “overexpressed”, i.e., expressedin increased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and direct methodsare preferred, for example, those involving SDS/PAGE and proteinstaining or western blotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein, polypeptide or peptide incomparison to the level in natural cells is indicative ofoverexpression, as is a relative abundance of the specific protein,polypeptides or peptides in relation to the other proteins produced bythe host cell and, e.g., visible on a gel.

In some embodiments, the expressed proteinaceous sequence forms aninclusion body in the host cell, the host cells are lysed, for example,by disruption in a cell homogenizer, washed and/or centrifuged toseparate the dense inclusion bodies and cell membranes from the solublecell components. This centrifugation can be performed under conditionswhereby the dense inclusion bodies are selectively enriched byincorporation of sugars, such as sucrose, into the buffer andcentrifugation at a selective speed. Inclusion bodies may be solubilizedin solutions containing high concentrations of urea (e.g. 8M) orchaotropic agents such as guanidine hydrochloride in the presence ofreducing agents, such as beta mercaptoethanol or DTT (dithiothreitol),and refolded into a more desirable conformation, as would be known toone of ordinary skill in the art.

VI. Immunological Compositions

In particular embodiments of the invention, immunological compositionsare employed. For the sake of brevity, the following section will referto any E. canis gp36 or E. chaffeensis gp47 immunological compositionsof the present invention, such as are described elsewhere herein as onlyexemplary embodiments. For example, the compositions may include all orpart of an E. canis gp36 SEQ ID NO:22, SEQ ID NO:37, SEQ ID NO:38, orSEQ ID NO:39. Also, the compositions may include all or part of an E.chaffeensis gp47 SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:40, or SEQ IDNO:41, for example. Antibodies may be utilized to bind an antigen,thereby rendering the molecule at least partially ineffective for itsactivity, for example. In other embodiments, antibodies to the antigenare employed in diagnostic aspects of the invention, such as fordetecting the presence of the antigen from a sample. Exemplary samplesmay be from an animal suspected of having E. canis or E. chaffeensisinfection, from an animal susceptible to E. canis or E. chaffeensisinfection, or from an animal that has an E. canis or E. chaffeensisinfection. Exemplary samples may be obtained from blood, serum,cerebrospinal fluid, urine, feces, cheek scrapings, nipple aspirate, andso forth.

Purified immunoreactive compositions or antigenic fragments of theimmunoreactive compositions can be used to generate new antibodies or totest existing antibodies (e.g., as positive controls in a diagnosticassay) by employing standard protocols known to those skilled in theart.

As is well known in the art, immunogenicity to a particular immunogencan be enhanced by the use of non-specific stimulators of the immuneresponse known as adjuvants. Exemplary and preferred adjuvants includecomplete BCG, Detox, (RIBI, Immunochem Research Inc.), ISCOMS andaluminum hydroxide adjuvant (Superphos, Biosector).

Included in this invention are polyclonal antisera generated by usingthe immunoreactive composition or a fragment of the immunoreactivecomposition as an immunogen in, e.g., rabbits. Standard protocols formonoclonal and polyclonal antibody production known to those skilled inthis art are employed. The monoclonal antibodies generated by thisprocedure can be screened for the ability to identify recombinantEhrlichia cDNA clones, and to distinguish them from known cDNA clones,for example.

The invention encompasses not only an intact monoclonal antibody, butalso an immunologically-active antibody fragment, e.g., a Fab or (Fab)2fragment; an engineered single chain scFv molecule; or a chimericmolecule, e.g., an antibody which contains the binding specificity ofone antibody, e.g., of murine origin, and the remaining portions ofanother antibody, e.g., of human origin.

In one embodiment, the antibody, or fragment thereof, may be linked to atoxin or to a detectable label, e.g. a radioactive label,non-radioactive isotopic label, fluorescent label, chemiluminescentlabel, paramagnetic label, enzyme label or colorimetric label. Examplesof suitable toxins include diphtheria toxin, Pseudomonas exotoxin A,ricin, and cholera toxin. Examples of suitable enzyme labels includemalate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,alcohol dehydrogenase, alpha glycerol phosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase,etc. Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, etc.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be usedaccording to the methods of this invention. There are numerous examplesof elements that are useful in magnetic resonance imaging. Fordiscussions on in vivo nuclear magnetic resonance imaging, see, forexample, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986)Magn. Reson. Med. 3, 336-340; Wolf, G. L., (1984) Physiol. Chem. Phys.Med. NMR 16, 93-95; Wesby et al., (1984) Physiol. Chem. Phys. Med. NMR16, 145-155; Runge et al., (1984) Invest. Radiol. 19, 408-415. Examplesof suitable fluorescent labels include a fluorescein label, anisothiocyalate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an opthaldehyde label, afluorescamine label, etc. Examples of chemiluminiscent labels include aluminal label, an isoluminal label, an aromatic acridinium ester label,a luciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of these and other suitablelabels, which may be employed in accordance with the present invention.The binding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedyet al., (1976) Clin. Chim. Acta 70, 1-31; and Schurs et al., (1977)Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in the laterare the glutaraldehyde method, the periodate method, the dimaleimidemethod, the maleimidobenzyl-N-hydroxy-succinimde ester method. All ofthese methods are incorporated by reference herein.

B. Antibodies

In certain aspects of the invention, one or more antibodies may beproduced to the expressed gp36 or gp47. These antibodies may be used invarious diagnostic and/or therapeutic applications described herein.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

“Mini-antibodies” or “minibodies” are also contemplated for use with thepresent invention. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992) Biochem 31:1579-1584. Theoligomerization domain comprises self-associating α-helices, e.g.,leucine zippers, that can be further stabilized by additional disulfidebonds. The oligomerization domain is designed to be compatible withvectorial folding across a membrane, a process thought to facilitate invivo folding of the polypeptide into a functional binding protein.Generally, minibodies are produced using recombinant methods well knownin the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B:120-126.

Antibody-like binding peptidomimetics are also contemplated in thepresent invention. Liu et al. Cell Mol Biol (Noisy-le-grand). 2003March; 49(2):209-16 describe “antibody like binding peptidomimetics”(ABiPs), which are peptides that act as pared-down antibodies and havecertain advantages of longer serum half-life as well as less cumbersomesynthesis methods.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will often be preferred.

However, “humanized” antibodies are also contemplated, as are chimericantibodies from mouse, rat, or other species, bearing human constantand/or variable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin.

C. Exemplary Methods for Generating Monoclonal Antibodies

Exemplary methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal witha LEE or CEE composition in accordance with the present invention andcollecting antisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art.Antibodies of the invention can also be produced transgenically throughthe generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or LEEs or CEEs encoding such adjuvants.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, N.J.),cytokines such as γ-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of polyclonal antibodies may be monitored by sampling bloodof the immunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60 61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen would occur at approximately two-weekintervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of ananimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender then incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83,1984). cites). For example, where the immunized animal is a mouse, onemay use P3 X63/Ag8, X63 Ag8.653, NS1/1.Ag 4 1, Sp210 Ag14, FO, NSO/U,MPC 11, MPC11 X45 GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3 Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICRLON HMy2 and UC729 6 are all useful in connection with human cellfusions. See Yoo et al., J Immunol Methods. 2002 Mar. 1; 261(1-2):1-20,for a discussion of myeloma expression systems.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the 8azaguanine resistant mouse murine myeloma SP2/0 non producer cell line.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp. 7174, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning and Microdroptechnology. The GS system is discussed in whole or part in connectionwith European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 andEuropean Patent Application No. 89303964.4.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the monoclonal antibodies soproduced by methods which include digestion with enzymes, such as pepsinor papain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonals. In one embodiment, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies. In another example, LEEs orCEEs can be used to produce antigens in vitro with a cell free system.These can be used as targets for scanning single chain antibodylibraries. This would enable many different antibodies to be identifiedvery quickly without the use of animals.

Another embodiment of the invention for producing antibodies accordingto the present invention is found in U.S. Pat. No. 6,091,001, whichdescribes methods to produce a cell expressing an antibody from agenomic sequence of the cell comprising a modified immunoglobulin locususing Cre-mediated site-specific recombination is disclosed. The methodinvolves first transfecting an antibody-producing cell with ahomology-targeting vector comprising a lox site and a targeting sequencehomologous to a first DNA sequence adjacent to the region of theimmunoglobulin loci of the genomic sequence which is to be converted toa modified region, so the first lox site is inserted into the genomicsequence via site-specific homologous recombination. Then the cell istransfected with a lox-targeting vector comprising a second lox sitesuitable for Cre-mediated recombination with the integrated lox site anda modifying sequence to convert the region of the immunoglobulin loci tothe modified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer, orby expression of full-length gene or of gene fragments in E. coli.

D. Antibody Conjugates

The present invention further provides antibodies against gp36 proteins,polypeptides and peptides, generally of the monoclonal type, that arelinked to at least one agent to form an antibody conjugate. In order toincrease the efficacy of antibody molecules as diagnostic or therapeuticagents, it is conventional to link or covalently bind or complex atleast one desired molecule or moiety. Such a molecule or moiety may be,but is not limited to, at least one effector or reporter molecule.Effector molecules comprise molecules having a desired activity, e.g.,cytotoxic activity. Non-limiting examples of effector molecules whichhave been attached to antibodies include toxins, anti-tumor agents,therapeutic enzymes, radio-labeled nucleotides, antiviral agents,chelating agents, cytokines, growth factors, and oligo- orpoly-nucleotides. By contrast, a reporter molecule is defined as anymoiety which may be detected using an assay. Non-limiting examples ofreporter molecules which have been conjugated to antibodies includeenzymes, radiolabels, haptens, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, luminescentmolecules, photoaffinity molecules, colored particles or ligands, suchas biotin.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an antibody conjugate. Such properties may beevaluated using conventional immunological screening methodology knownto those of skill in the art. Sites for binding to biological activemolecules in the antibody molecule, in addition to the canonical antigenbinding sites, include sites that reside in the variable domain that canbind pathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic or anticellular agent, and may be termed “immunotoxins”.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody directed imaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, idoine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium 186, rhenium 188, ⁷⁵selenium, ³⁵sulphur, technicium99m and/oryttrium⁹⁰. ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present invention may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the invention may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column. Alternatively, directlabeling techniques may be used, e.g., by incubating pertechnate, areducing agent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and/or avidinand streptavidin compounds. The use of such labels is well known tothose of skill in the art and are described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6 α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In another embodiment of the invention, the anti-gp36 antibodies arelinked to semiconductor nanocrystals such as those described in U.S.Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all ofwhich are incorporated herein in their entireties); as well as PCTPublication No. 99/26299 (published May 27, 1999). In particular,exemplary materials for use as semiconductor nanocrystals in thebiological and chemical assays of the present invention include, but arenot limited to those described above, including group II-VI, III-V andgroup IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP,GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si andternary and quaternary mixtures thereof. Methods for linkingsemiconductor nanocrystals to antibodies are described in U.S. Pat. Nos.6,630,307 and 6,274,323.

E. Immunodetection Methods

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such asimmunoreactive polypeptides. The antibodies prepared in accordance withthe present invention may be employed to detect wild type and/or mutantproteins, polypeptides and/or peptides. The use of wild-type and/ormutant antibodies is contemplated. Some immunodetection methods includeenzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay,bioluminescent assay, and Western blot to mention a few. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Doolittle MH and Ben-Zeev O, 1999;Gulbis B and Galand P, 1993; De Jager R et al., 1993; and Nakamura etal., 1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of comprising protein, polypeptide and/or peptide, andcontacting the sample with a first anti-gp36 (or gp47) antibody inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of immunocomplexes.

These methods include methods for purifying wild type and/or mutantproteins, polypeptides and/or peptides as may be employed in purifyingwild type and/or mutant proteins, polypeptides and/or peptides frompatients' samples and/or for purifying recombinantly expressed wild typeor mutant proteins, polypeptides and/or peptides. In these instances,the antibody removes the antigenic wild type and/or mutant protein,polypeptide and/or peptide component from a sample. The antibody willpreferably be linked to a solid support, such as in the form of a columnmatrix, and the sample suspected of containing the wild type or mutantprotein antigenic component will be applied to the immobilized antibody.The unwanted components will be washed from the column, leaving theantigen immunocomplexed to the immobilized antibody, which wild type ormutant protein antigen is then collected by removing the wild type ormutant protein and/or peptide from the column.

The immunobinding methods also include methods for detecting andquantifying the amount of a wild type or mutant protein reactivecomponent in a sample and the detection and quantification of any immunecomplexes formed during the binding process. Here, one would obtain asample suspected of comprising a wild type or mutant protein and/orpeptide or suspected of comprising an E. canis organism, and contact thesample with an antibody against wild type or mutant, and then detect andquantify the amount of immune complexes formed under the specificconditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing a wild type or mutantprotein-specific antigen, such as a specimen, a homogenized tissueextract, a cell, separated and/or purified forms of any of the abovewild type or mutant protein-containing compositions, or even anybiological fluid that comes into contact with an E. canis organism uponinfection.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any proteinantigens present. After this time, the sample-antibody composition, suchas a tissue section, ELISA plate, dot blot or western blot, willgenerally be washed to remove any non-specifically bound antibodyspecies, allowing only those antibodies specifically bound within theprimary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241, each incorporated herein by reference. Ofcourse, one may find additional advantages through the use of asecondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection uses two different antibodies. A firststep biotinylated, monoclonal or polyclonal antibody is used to detectthe target antigen(s), and a second step antibody is then used to detectthe biotin attached to the complexed biotin. In that method the sampleto be tested is first incubated in a solution containing the first stepantibody. If the target antigen is present, some of the antibody bindsto the antigen to form a biotinylated antibody/antigen complex. Theantibody/antigen complex is then amplified by incubation in successivesolutions of streptavidin (or avidin), biotinylated DNA, and/orcomplementary biotinylated DNA, with each step adding additional biotinsites to the antibody/antigen complex. The amplification steps arerepeated until a suitable level of amplification is achieved, at whichpoint the sample is incubated in a solution containing the second stepantibody against biotin. This second step antibody is labeled, as forexample with an enzyme that can be used to detect the presence of theantibody/antigen complex by histoenzymology using a chromogen substrate.With suitable amplification, a conjugate can be produced which ismacroscopically visible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as variousforms of hyperproliferative diseases, such as cancer, includingleukemia, for example. Here, a biological and/or clinical samplesuspected of containing a wild type or mutant protein, polypeptide,peptide and/or mutant is used. However, these embodiments also haveapplications to non-clinical samples, such as in the titering of antigenor antibody samples, for example in the selection of hybridomas.

F. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

In one exemplary ELISA, the antibodies of the invention are immobilizedonto a selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the wild type and/or mutant protein antigen, such as aclinical sample, is added to the wells. After binding and/or washing toremove non-specifically bound immune complexes, the bound wild typeand/or mutant protein antigen may be detected. Detection is generallyachieved by the addition of another antibody that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA”.Detection may also be achieved by the addition of a second antibody,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label.

In another exemplary ELISA, the samples suspected of containing the wildtype and/or mutant protein antigen are immobilized onto the well surfaceand/or then contacted with the antibodies of the invention. Afterbinding and/or washing to remove non-specifically bound immunecomplexes, the bound antibodies are detected. Where the initialantibodies are linked to a detectable label, the immune complexes may bedetected directly. Again, the immune complexes may be detected using asecond antibody that has binding affinity for the first antibody, withthe second antibody being linked to a detectable label.

Another ELISA in which the wild type and/or mutant proteins,polypeptides and/or peptides are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst wild type or mutant protein are added to the wells, allowed tobind, and/or detected by means of their label. The amount of wild typeor mutant protein antigen in an unknown sample is then determined bymixing the sample with the labeled antibodies against wild type and/ormutant before and/or during incubation with coated wells. The presenceof wild type and/or mutant protein in the sample acts to reduce theamount of antibody against wild type or mutant protein available forbinding to the well and thus reduces the ultimate signal. This is alsoappropriate for detecting antibodies against wild type or mutant proteinin an unknown sample, where the unlabeled antibodies bind to theantigen-coated wells and also reduces the amount of antigen available tobind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

G. Immunohistochemistry

The antibodies of the present invention may also be used in conjunctionwith both fresh-frozen and/or formalin-fixed, paraffin-embedded tissueblocks prepared for study by immunohistochemistry (IHC). The method ofpreparing tissue blocks from these particulate specimens has beensuccessfully used in previous IHC studies of various prognostic factors,and/or is well known to those of skill in the art (Brown et al., 1990;Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” tissue at room temperature in phosphate buffered saline(PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and/or pelleting again by centrifugation;snap-freezing in 70° C. isopentane; cutting the plastic capsule and/orremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and/or cutting 25-50 serial sections.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and/or embedding the block in paraffin; and/or cutting upto 50 serial permanent sections.

H. Immunoelectron Microscopy

The antibodies of the present invention may also be used in conjunctionwith electron microscopy to identify intracellular tissue components.Briefly, an electron-dense label is conjugated directly or indirectly tothe antibody. Examples of electron-dense labels according to theinvention are ferritin and gold. The electron-dense label absorbselectrons and can be visualized by the electron microscope.

I. Immunodetection Kits

In still further embodiments, the present invention concernsimmunodetection kits for use with the immunodetection methods describedabove. As the antibodies are generally used to detect wild type and/ormutant proteins, polypeptides and/or peptides, the antibodies willpreferably be included in the kit. However, kits including both suchcomponents may be provided. The immunodetection kits will thus comprise,in suitable container means, a first antibody that binds to a wild typeand/or mutant protein, polypeptide and/or peptide, and/or optionally, animmunodetection reagent and/or further optionally, a wild type and/ormutant protein, polypeptide and/or peptide.

In preferred embodiments, monoclonal antibodies will be used. In certainembodiments, the first antibody that binds to the wild type and/ormutant protein, polypeptide and/or peptide may be pre-bound to a solidsupport, such as a column matrix and/or well of a microtitre plate.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with and/orlinked to the given antibody. Detectable labels that are associated withand/or attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and/or all such labels may beemployed in connection with the present invention.

The kits may further comprise a suitably aliquoted composition of thewild type and/or mutant protein, polypeptide and/or polypeptide, whetherlabeled and/or unlabeled, as may be used to prepare a standard curve fora detection assay. The kits may contain antibody-label conjugates eitherin fully conjugated form, in the form of intermediates, and/or asseparate moieties to be conjugated by the user of the kit. Thecomponents of the kits may be packaged either in aqueous media and/or inlyophilized form.

The container means of the kits will be suitable housed and willgenerally include at least one vial, test tube, flask, bottle, syringeand/or other container means, into which the antibody may be placed,and/or preferably, suitably aliquoted. Where wild type and/or mutantgp36 protein, polypeptide and/or peptide, and/or a second and/or thirdbinding ligand and/or additional component is provided, the kit willalso generally contain a second, third and/or other additional containerinto which this ligand and/or component may be placed. The kits of thepresent invention will also typically include a means for containing theantibody, antigen, and/or any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionand/or blow-molded plastic containers into which the desired vials areretained.

VII. Pharmaceutical Preparations

It is also contemplated that pharmaceutical compositions may be preparedusing the novel compositions of the present invention. In such a case,the pharmaceutical composition comprises the novel active composition ofthe present invention and a pharmaceutically acceptable carrier. Aperson having ordinary skill in this art would readily be able todetermine, without undue experimentation, the appropriate dosages androutes of administration of the active component of the presentinvention.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a subject. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

In general, a pharmaceutical composition of the present invention maycomprise an E. canis gp36 polypeptide, polynucleotide, or antibodyand/or an E. chaffeensis gp47 polypeptide, polynucleotide, or antibody,and/or mixtures thereof

A protein may be formulated into a composition in a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids such as acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media, which can be employed, will be knownto those of skill in the art in light of present disclosure. Forexample, one dosage could be dissolved in lmL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more agents that target a polypeptide or thesecretion thereof or additional agent dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical,”“pharmaceutically acceptable,” or “pharmacologically acceptable” refersto molecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one agent that targetsthe polypeptide or the secretion thereof and/or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The invention may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g. aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof

The invention may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the composition is prepared for administration bysuch routes as oral ingestion. In these embodiments, the solidcomposition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations that are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

VIII. Exemplary Kits of the Invention

In particular embodiments of the invention, there is a kit housed in asuitable container. The kit may be suitable for diagnosis, treatment,and/or protection for an individual from Ehrlichia, such as Ehrlichiacanis, Ehrlichia chaffeensis, or both. In particular embodiments, thekit comprises in a suitable container an agent that targets an E. canisgp36 antigen or an E. chaffeensis gp47 antigen. The agent may be anantibody, a small molecule, a polynucleotide, a polypeptide, a peptide,or a mixture thereof. The agent may be provided in the kit in a suitableform, such as sterile, lyophilized, or both, for example. In particularembodiments, the kit comprises one or more of the following: 1) anantibody against one or more of SEQ ID NO:22, SEQ ID NO:37, SEQ IDNO:38, or SEQ ID NO:39 (for E. canis); 2) an antibody against one ormore of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:40, or SEQ ID NO:41 (forE. chaffeensis); and/or 3) SEQ ID NO:22, SEQ ID NO:37, SEQ ID NO:38, orSEQ ID NO:39 (for E. canis) and/or SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:40, or SEQ ID NO:41 (for E. chaffeensis) and/or related proteinsthereof. Other E. canis gp36-related or E. chaffeensis gp47-relatedimmunogenic-related compositions (including polypeptides, peptides, orantibodies) not specifically presented herein may also be included.

The kit may further comprise one or more apparatuses for delivery of acomposition to an individual in need thereof. The apparatuses mayinclude a syringe, eye dropper, needle, biopsy tool, scoopula, catheter,and so forth, for example.

In embodiments wherein the kit is employed for a diagnostic purpose, thekit may further provide one or more detection compositions orapparatuses for identifying an E. canis gp36 antigen, an E. chaffeensisgp47 antigen, or both. Such an embodiment may employ a detectable label,such as for an antibody, for example, and the label may be fluorescent,chemiluminescent, or colorimetric, for example.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exemplary Materials and Methods

The following descriptions provide merely exemplary materials andmethods utilized in the invention.

Ehrlichiae and Purification.

E. canis (Jake, Oklahoma, and Demon isolates) and E. chaffeensis(Arkansas and Sapulpa isolates) were propogated as previously described(McBride et al., 2001). Ehrlichiae were purified by size exclusion overSephacryl S-1000 (Amersham Biosciences, Piscataway, N.J.) as previouslydescribed (Rikihisa et al., 1992). The fraction containing bacteria wasfrozen and utilized as an antigen and DNA source.

Construction and Screening of the E. canis Genomic Library.

An E. canis HpaII genomic library was constructed and screened aspreviously described (McBride et al., 2001).

DNA Sequencing.

Library inserts, plasmids, and PCR products were sequenced with an ABIPrism 377XL DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City,Calif.) at the University of Texas Medical Branch Protein Chemistry CoreLaboratory.

Glycoprotein Polynucleotide Analysis.

Nucleic and amino acid alignments were performed with MegAlign(Lasergene v5.08, DNAstar, Madison, Wis.). The gp36 and gp47 proteinsequences were tested for potential mucin-type O-linked glycosylation onserines and threonines with the computational algorithm NetOGlyc(Julenius et al., 2005). The tandem repeats of the genes encoding gp36of E. canis strains Jake, Oklahoma, and Demon; gp47 of E. chaffeensisstrains Arkansas and Sapulpa; mucin-like proteins of E. ruminantiumstrains Highway (AF308673; SEQ ID NO:1, at least part of which encodesAAL08844 (SEQ ID NO:31)), Welgevonden (the genome is provided in GenBankAccession No. CR767821; an exemplary mucin-like protein thereof isprovided in GenBank Accession No. CAI26602 (SEQ ID NO:29)), and Gardel(the genome is provided in GenBank Accession No. CR925677; an exemplarymucin-like protein thereof is provided in GenBank Accession No. CAI27556(SEQ ID NO:30)); gp140 of E. canis strain Jake (AF112369; SEQ ID NO:2),and gp120 of E. chaffeensis strains Arkansas (ECU49426; SEQ ID NO:3) andSapulpa (ECU74670; SEQ ID NO:4) were analyzed by the Tandem RepeatFinder (Benson, 1999) for period size, number of repeats, and percenthomology between the repeats. The gp36 and gp47 protein sequences weretested for the presence of signal sequences with the computationalalgorithm SignalP trained on gram-negative bacteria (Nielsen et al.,1997).

PCR amplification of the Ehrlichia glycoprotein genes. Primers for theamplification of the E. canis and E. chaffeensis gp36 and gp47 geneswere designed using Primer Select (Lasergene v5.08, DNAstar, MadisonWis.). Primers corresponding to nucleotides 28 to 47 (5′-ATG CTT CAT TTAACA ACA GA, forward; SEQ ID NO:5) and 794 to 816 within the ORF (5′-AGAATC TAA ATC TAA AAG TCC AG, reverse; SEQ ID NO:6) were used to amplifythe E. canis gp36 gene. E. canis DNA was amplified using the PCR Mastermix (F. Hoffmann-La Roche Ltd, Basel, Switzerland) with a thermalcycling profile of 95° C. for 4 min and 30 cycles of 95° C. for 30 s,55° C. for 30 s, and 72° C. for 1 min, followed by a 72° C. extensionfor 7 min and a 4° C. hold. PCR products were separated in 1% agarosegels. Primers corresponding to nucleotides 4 to 22 (5′-CTT CAT TTA ACAACA GAA A, forward; SEQ ID NO:7) and 902 to 924 within the ORF (5′-TTGAGC AGC CAT ATC TTC TTC AT, reverse; SEQ ID NO:8) were used to amplifythe E. chaffeensis gp47 gene using the same PCR conditions. Recombinantprotein containing the amino-terminus of E. canis gp36 was created byamplifying respective DNA with primers corresponding with nucleotides 28to 47 (5′-ATG CTT CAT TTA ACA ACA GA, forward; SEQ ID NO:9) andnucleotides 321 to 345 (5′-TTG ATA AGC ATG CAC AGA AAT AAA G, reverse;SEQ ID NO:10), and the carboxyl-terminus was amplified with primersspecific for nucleotides 370-392 (5′-GGA AAT CCA TCA CGT CCT GCT AT,forward; SEQ ID NO:11) and 794 to 816 (5′-AGA ATC TAA ATC TAA AAG TCCAG, reverse; SEQ ID NO:12). Recombinant protein containing theamino-terminus of E. chaffeensis gp47 was created by amplifyingrespective DNA with primers corresponding with nucleotides 4 to 22(5′-CTT CAT TTA ACA ACA GAA A, forward; SEQ ID NO:13) and nucleotides436 to 459 (5′-AAC TGG AAC CAC TAT ACT GTC ACT, reverse; SEQ ID NO:14)and the carboxyl-terminus was amplified with primers specific fornucleotides 439-463 (5′-GAC AGT ATA GTG GTT CCA GTT CTT G, forward; SEQID NO:15) and 902 to 924 (5′-TTG AGC AGC CAT ATC TTC TTC AT, reverse;SEQ ID NO:16).

Cloning and Expression of Recombinant Ehrlichia Glycoproteins.

The amplified PCR product was cloned directly into the pBAD Thio TOPO®expression vector (Invitrogen, Carlsbad, Calif.). TOP10 E. coli(Invitrogen) were transformed with the plasmid containing the E. canisgp36 or E. chaffeensis gp47 genes, and positive transformants werescreened by PCR for the presence of the insert and orientation andsequenced to confirm the reading frame of the genes. Recombinant proteinexpression was induced with 0.2% arabinose for 3 h at 37° C. Bacteriawere pelleted (5,000×g for 20 min), resuspended in PBS, and recombinantproteins were purified under native conditions as previously described(Doyle et al., 2005).

Gel Electrophoresis and Western Immunoblotting.

Purified E. canis or E. chaffeensis antigens were separated by SDS-PAGE,transferred to nitrocellulose, and Western blots performed as previouslydescribed (McBride et al., 2003), except primary antibodies were diluted(1:500). Sera from HME patients were a kind gift from Focus Technologies(Cypress, Calif.).

Mouse Immunization.

Five BALB/c mice (Jackson Laboratories, Bar Harbor, Me.) were immunizedwith the recombinant E. canis gp36 or E. chaffeensis gp47 proteins.Recombinant protein (100 μg) in 0.1 mL was mixed with an equal volume ofFreund's complete adjuvant (Sigma, St. Louis, Mo.) for the firstinjection and with Freund's incomplete adjuvant for the subsequentinjections. The mice were given intraperitoneal injections twice at twoweek intervals.

Recombinant Fusion Proteins.

Two 27-bp complementary oligonucleotides (Sigma-Genosys, Woodlands,Tex.) encoding a 9-mer repeat region of E. canis gp36 were synthesized.The coding strand contained additional 5′ nucleotides CACC fordirectional TOPO vector cloning (5′-CACC ACT GAA GAT TCT GTT TCT GCT CCAGCT (SEQ ID NO:17; reverse complement 5′-AGC TGG AGC AGA AAC AGA ATC TTCAGT; SEQ ID NO:18). The oligos were resuspended in water (200 μM),combined and diluted to 100 μM in oligonucleotide annealing buffer (10mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA), then heated to 95° C. for15 min and allowed to slowly cool to room temperature. This mixture wassubsequently used for standard cloning into the pBAD Directional TOPO®Expression vector (Invitrogen) to express the 9-mer, TEDSVSAPA (SEQ IDNO:22), as a thioredoxin fusion protein. This procedure was repeatedwith the 17-mer repeat unit of the E. chaffeensis gp47, (oligo sequences5′-CACC GCT AGT GTA TCT GAA GGA GAT GCA GTA GTA AAT GCT GTA AGC CAA GAAACT CCT GCA (SEQ ID NO:19); reverse complement 5′-TGC AGG AGT TTC TTGGCT TAC AGC ATT TAC TAC TGC ATC TCC TTC AGA TAC ACT AGC; SEQ ID NO:20)).

Enzyme-Linked Immunosorbent Assay (ELISA).

ELISA plates (Nunc-Immuno™ Plates with MaxiSorp™ Surface, NUNC,Roskilde, Denmark) were coated with protein or peptide (2 μg/well, 100μL) in phosphate buffered saline (PBS). Periodate treatment of therecombinant repeat fusion protein was carried out for 20 min in 100 mMsodium acetate/5 mM EDTA buffer with 10 mM sodium metaperiodate. Antigenwas absorbed to the ELISA plates overnight at 4° C. or for 2 hr at roomtemperature with gentle agitation and subsequently washed three timeswith TBS-Tween 20 (300 μL), blocked with 1× milk diluent/blockingsolution (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) for 1 hrat 37° C. with agitation and washed again. Anti-E. canis sera diluted(1:500) in milk diluent was added to each well (100 μL) and incubated atroom temperature for 1.5 h with gentle agitation. The plates were washedfour times and an alkaline phosphatase-labeled goat anti-dog IgG (H+L)secondary antibody (1:3000) (Kirkegaard & Perry Laboratories) in milkdiluent was added and incubated for 1 hr. The plates were washed fourtimes and substrate (100 μL) (BluePhos, Kirkegaard & Perry Laboratories)was added to each well. The plates were incubated for 30 min in the darkwith agitation, and color development was stopped with 1% SDS. Theplates were subsequently read on a microplate reader (Versamax,Molecular Devices, Sunnyvale, Calif.) at A650 and data analyzed bySoftmaxPro v4.0 (Molecular Devices). The data represents the mean ofthree independent dog sera. A 20-mer p28-19 VR1 peptide, sequenceNH2-RNTTVGVFGLKQNWDGSAIS (SEQ ID NO:21) (a kind gift from Dr. X. J. Yu),was used as a positive control peptide to confirm binding andimmunoreactivity.

Immunoelectron Microscopy.

Immunogold electron microscopy was performed as previously described(Doyle et al., 2005).

Analysis of Secreted Immunoreactive Proteins.

E. canis or E. chaffeensis infected DH82 cells were monitored until90-100% of the monolayer cells were infected. Three days prior tosupernatant harvest, the culture medium (DMEM supplemented with 10%bovine calf serum) was completely removed and replaced with serum-freeDMEM. Culture supernatants were collected without disturbing the cellmonolayer and centrifuged (5000×g for 20 min) to pellet cells andbacteria. Supernatants were subsequently concentrated 40-fold (AmiconUltra Centifugal Filter Devices with a 10-kDa MW cutoff; Millipore,Billerica, Mass.). Cell culture supernatants (2 μL) were diluted 1:2 inLDS sample buffer, separated by gel electrophoresis, transferred tonitrocellulose and immunoreactive proteins detected by Westernimmunoblotting using anti-E. canis polyclonal antibody (1:500) asdescribed previously.

Indirect Fluorescent Antibody Analysis (IFA) and Confocal Microscopy.

Antigen slides were prepared from DH82 cells infected with E. canis(Jake isolate) or E. chaffeensis (Arkansas isolate) as describedpreviously (McBride et al., 2001). Monospecific rabbit serum producedagainst the recombinant E. canis disulfide bond formation protein (DsbA)(McBride et al. 2002) was diluted 1:100 and added to each well (15 μL)and allowed to incubate for 30 min. Slides were washed, and either mouseanti-gp36 or mouse anti-gp47 (1:100 dilution) was added and incubatedfor 30 min. Alexa Fluor® 488 goat anti-rabbit IgG (H & L) secondaryantibody (Molecular Probes, Eugene, Oreg.) diluted 1:100 was added andincubated for 30 min, followed by washing and subsequent addition andincubation of rhodamine-labeled goat anti-mouse IgG (H & L) secondaryantibody (Kirkegaard & Perry Laboratories). Slides were viewed in theOptical Imaging Laboratory at UTMB using a Zeiss LSM-510 META confocalmicroscope.

Nucleotide Sequence Accession Numbers.

The E. canis gp36 gene sequences from the Jake, Oklahoma, and Demonisolates and E. chaffeensis gp47 gene sequences from the Arkansas andSapulpa isolates were deposited into GenBank and assigned the followingaccession numbers, respectively: E. canis Jake (DQ085427; SEQ ID NO:32,which encodes SEQ ID NO:37), E. canis Oklahoma (DQ085428; SEQ ID NO:33,which encodes SEQ ID NO:38), E. canis Demon (DQ085429; SEQ ID NO:34,which encodes SEQ ID NO:39), E. chaffeensis Arkansas (DQ085430; SEQ IDNO:35, which encodes SEQ ID NO:40), E. chaffeensis Sapulpa (DQ085431;SEQ ID NO:36, which encodes SEQ ID NO:41).

Exemplary “mucin” polynucleotides from Highway (SEQ ID NO:42),Welgevondon (SEQ ID NO:43) and Gardel (SEQ ID NO:44) E. ruminantiumstrains are orthologs of gp36 and gp47.

Example 2 Molecular Identification of the E. Canis 37-kDa MajorImmunoreactive Protein

A positive clone with a 1.5-kb insert contained a complete open readingframe (ORF) encoding a predicted protein with a molecular mass of29.3-kDa (26.7 without predicted 23 amino acid signal peptide) withhomology to highly glycosylated eukaryotic mucin proteins. With theprevious correlation of glycoproteins with major immunoreactiveproteins, this candidate was of particular interest. The gene containedtwelve tandem repeats at the 3′ end of the ORF (FIG. 1) encoding 9 aminoacids (Table 2). The NetOGlyc O-linked glycosylation prediction serverpredicted that the serines and threonines of the tandem repeats, withexemplary predicted sequence TEDSVSAPA (SEQ ID NO:22), were the sites ofglycosylation (bold letters, Table 1). The E. chaffeensis Arkansasstrain genome was BLAST searched with the E. canis gp36 sequence, and ahomologous open reading frame encoding a protein with seven exemplary19-mer tandem repeats (ASVSEGDAVVNAVSQETPA; SEQ ID NO:23) and apredicted mass of 32.9-kDa was identified. The DNA sequence upstream ofthe tandem repeat region contained a similarity index of 61.5% (57.0%amino acid), but tandem repeat regions were not homologous.

TABLE 2 Ehrlichia tandem repeats of major immunoreactive glycoproteins.Repeat Repeat length Copy number percent Consensus Tandem RepeatSequence Source Strain (bp) (DNA seq) homology (amino acid) Eca gp36Jake 27 12.2 100 TEDSVSAPA (SEQ ID NO: 22) Oklahoma 27 5.2 100 Demon 2716.2 100 Ech gp47 Arkansas 57 7.0 99 ASVSEGDAVVNAVSQETPA (SEQ ID NO: 23)Sapulpa 99 4.5 99 EGNASEPVVSQEAAPVSESGDAANPVSSSENAS (SEQ ID NO: 24) EruHighway 27 21.7 99 VTSSPEGSV (SEQ ID NO: 25) Mucin-like Welgevonden 2756.0 95 protein Gardel 66 16.9 99 SSEVTESNQGSSASVVGDAGVQ (SEQ ID NO: 26)Eca Jake 108 14.3 96 KEESTPEVKAEDLQPAVDGSVEHSSSEVGEKVSETS gp140 (SEQ IDNO: 27) Ech Arkansas 240 4.5 98 EDEIVSQPSSEPFVAESEVSKVEQEETNPEVLIKDLQgp120 Sapulpa 240 3.5 97 DVASHESGVSDQPAQVVTERESEIESHQGETEKESG ITESHQK(SEQ ID NO: 28)

The genes encoding gp36 of E. canis Oklahoma and Demon strains as wellas the gp47 of E. chaffeensis Sapulpa strain were sequenced to identifypotential variations in the gene sequence. Different E. canis strainsretained identical tandem repeat sequence, but they differed in thenumber of the repeats (Table 2). Interestingly, the Sapulpa strain of E.chaffeensis encoded an entirely different set of tandem repeats (4 fullrepeats of 33 amino acids) than found in E. chaffeensis Arkansas strain.The E. chaffeensis gp47 gene sequences upstream of the repeat regioncontained 99.8% homology between strains, but had a low degree of(27.7%) homology primarily associated with the 3′ region downstream ofthe tandem repeats. The region just upstream of the Arkansas tandemrepeats encodes the amino acids Glu-Gly-Asn, which are the 1st threeamino acids of the Sapulpa strain repeat, suggesting a more recenttandem repeat switch. The nucleic acid sequence within the tandemrepeats of each gp47 gene was highly conserved (at least 99%) (Table 2).

Although the tandem repeat sequences varied greatly among the differentspecies and strains, there was a conservation of amino acid usage amongthe repeats. A total of ten amino acids were used in the all of therepeats, with a particularly high occurrence of serine, threonine,alanine, proline, valine, and glutamic acid. Analysis of theglycoprotein amino acid sequence upstream of the repeats compared tothat including the repeats until the termination codon demonstrated asubstantial increase in usage of these amino acids (Table 3). Predictedglycosylation sites by NetOGlyc were found only within the tandemrepeats of the proteins (Table 2). The threonine residues within the E.chaffeensis repeat were predicted sites for glycan attachment, andserine residues exhibited a high potential, but were not identified asglycan attachment sites. Similarly, the E. ruminantium Gardel strain“mucin-like” protein contained a threonine and several serine residuesthat were slightly below the predicted threshold as sites of glycanattachment.

TABLE 3 Amino acid analysis of ehrlichial glycoproteins. Ser Thr Rpt-Rpt- Ala Pro Val Glu Source Strain Non-rpt term Non-rpt term Non-rptRpt-term Non-rpt Rpt-term Non-rpt Rpt-term Non-rpt Rpt-term Eca gp36Jake 6.4 22.0 4.1 11.9 5.3 22.0 4.1 11.0 5.3 11.0 3.5 11.0 Oklahoma 6.421.7 4.1 13.0 5.3 21.7 4.1 10.9 5.3 10.9 3.5 10.9 Demon 6.4 22.1 4.111.7 5.3 22.1 4.1 11.0 5.3 11.0 3.5 11.0 Ech gp47 Arkansas 9.2 15.8 3.34.5 6.0 21.8 3.3 5.3 8.7 21.1 5.4 10.5 Sapulpa 9.9 21.8 3.3 1.4 6.0 17.03.3 9.5 8.8 12.2 5.0 15.7 Eru Mucin- Highway 8.9 32.3 6.4 11.3 2.6 1.01.9 11.3 8.3 21.5 6.4 11.3 like protein Welgevonden 8.2 27.2 6.3 11.11.9 12.3 1.9 11.1 7.6 15.3 7.6 11.1 Gardel 8.8 27.4 5.0 4.6 2.5 8.3 1.90 7.6 18.8 7.6 9.1

Example 3 Immunoreactivity and Glycosylation of gp36 and gp47

The recombinant E. canis gp36 reacted strongly with serum antibodiesfrom a dog experimentally infected with E. canis (FIG. 2A, lane 1).Following cleavage of the fusion partner thioredoxin, the recombinantprotein exhibited a molecular mass of 36-kDa (data not shown), which wassignificantly larger than predicted by amino acid sequence (26.7 kDa).Carbohydrate was detected on the recombinant gp36 (FIG. 2A, lane 2). Therecombinant E. chaffeensis gp47 also exhibited strong immunoreactivity(FIG. 2B, lane 1), migrated larger than the predicted mass (32.9 kDa),and carbohydrate was detected (FIG. 2B, lane 2).

Example 4 Identification of Native gp36 and gp47

A native E. canis protein of molecular mass 36-kDa, which correspondedto the ˜37-kDa protein previously described (McBride et al., 2003),reacted with monospecific mouse antiserum produced against therecombinant protein by Western blot (FIG. 3A). A less prominent proteinwas also visualized at 34-kDa. Mouse anti-recombinant gp47 identified a47-kD protein in E. chaffeensis whole cell lysates (FIG. 3B). Westernimmunoblots of E. chaffeensis whole cell lysates were reacted with tensuspected HME patient sera that had detectable E. chaffeensis antibodiesby indirect fluorescent antibody analysis (IFA). Seven of ten serarecognized an immunoreactive 47-kDa protein identical in mass to theprotein recognized by anti-recombinant gp47 serum (FIG. 3B).

Example 5 Early Antibody Response to gp36

Kinetic studies of the host response to E. canis demonstrated that a˜37-kDa antigen was recognized earliest by antibodies in acute phasesera from dogs experimentally infected with E. canis (McBride et al.,2003). Western immunoblot confirmed that the recombinant gp36 was notrecognized by pre-inoculation sera, but that antibodies were producedagainst gp36 in the early acute phase (day 14) (FIG. 4), confirming theidentity of the gp36 as the major 37-kDa antigen of E. canis. Theantibody response against gp36 remained very strong throughconvalescence (day 56) (FIG. 4).

Example 6 Immunoreactivity of the gp36 and gp47 Tandem Repeats

Western immunoblotting determined that the carboxyl-terminus includingthe tandem repeats was the highly immunoreactive portion of the protein,but homologous N-terminal regions preceeding the tandem repeat regionsof the gp36 and gp47 were not immunoreactive (data not shown). A singlerepeat expressed as a recombinant fusion protein was recognized byanti-E. canis dog serum (FIG. 5B). The fusion protein containing the9-mer demonstrated an electrophoretic shift larger than the predictedmass (˜900 kDa), suggesting that the peptide was post-translationallymodified and corroborating the NetOGlyc prediction, that identified therepeat units as sites of glycan attachment (FIG. 5A). A fusion proteincontaining a single 17-mer repeat of the E. chaffeensis gp47 was alsorecognized by anti-E. chaffeensis dog serum (FIG. 5C). The gp36 and gp47were antigenically distinct, as neither reacted with heterologousantisera (FIGS. 6A and 6B).

Carbohydrate is an important part of the gp36 epitope determinant. Aseach tandem repeat unit of E. canis gp36 was a predicted site ofglycosylation and found to contain a major B cell epitope, the presentinventors hypothesized that attached glycans were important epitopedeterminants. To confirm this, the 9-mer repeat fusion protein wastreated with periodate to test antibody recognition following structuralmodification of the glycan. The untreated 9-mer fusion protein reactedwith anti-E. canis dog serum by ELISA; however, the recognition ofperiodate treated 9-mer fusion protein fusion was greatly reduced and asynthetic peptide with the sequence of the repeat region was notrecognized at all, demonstrating the requirement of post-translationalmodification for antibody binding (FIG. 6). Structural modification ofthe glycan by periodate treatment reduced antibody recognition of theepitope almost to the background level of thioredoxin alone asdetermined by reduction in ELISA O.D. values.

Example 7 Carbohydrate is an Important gp36 Epitope Determinant

As each tandem repeat unit of E. canis gp36 was a predicted site ofglycosylation and found to contain a major B-cell epitope, the inventorsconsidered that attached glycans were important epitope determinants. Tocharacterize this, recombinant E. canis gp36 was treated with periodateto test antibody recognition following structural modification of theglycan. The sham-treated gp36 reacted strongly with anti-E. canis dogserum by ELISA, while the periodate-treated recombinant gp36 wassubstantially reduced (FIG. 7A). To further characterize thisobservation, ELISA was used to test the recognition of recombinantfusion proteins with a single repeat (9-mer; TEDSVSAPA; SEQ ID NO:22), a12-mer (SVSAPATEDSVS; SEQ ID NO:45), and two tandem repeat units(18-mer; TEDSVSAPATEDSVSAPA; SEQ ID NO:46) from gp36 in comparison withnonglycosylated synthetic peptides with the identical sequences. Whereasall of the recombinant proteins were recognized with immune dog serum,the synthetic single repeat (9-mer) was not recognized at all, and theoverlapping peptide (12-mer) exhibited minimal reactivity, demonstratingthe importance of posttranslational modification for antibody binding tothese peptides (FIG. 7B). The tandem repeat (18-mer) synthetic peptidewas recognized by dog serum, although not as well as the recombinant,demonstrating the presence of a linear amino acid-based epitope presentin a tandem repeat unit-containing peptide (18-mer) (FIG. 7B).Recognition of the recombinant E. chaffeensis repeat fusion proteinexhibited an absorbance by ELISA higher than that of synthetic peptide(FIG. 7C).

Example 8 Cellular Localization and Secretion of gp36 and gp47

Ehrlichiae exist in two distinct morphologic forms known as reticulateand dense-cored (Popov et al., 1995). The localization of E. canis gp36(FIG. 8A) and E. chaffeensis gp47 (FIG. 8B) by immunogold electronmicroscopy found that these proteins were differentially expressed onthe surface of the dense-cored form of the bacteria, but not thereticulate form. The gp36 and gp47 were also associated with the morulamembranes containing the dense-cored morphological forms of the bacteria(FIGS. 8A and 8B). Differential expression of the E. canis gp36 and E.chaffeensis gp47 was further confirmed by IFA analysis and confocalmicroscopy of infected cell slides using antibodies against theconserved ehrlichial disulfide bond formation protein Dsb in addition togp36 or gp47. The IFA demonstrated that E. canis gp36 and E. chaffeensisgp47 were expressed on a subset of ehrlichiae compared to the expressionof Dsb (constitutively expressed).

The E. canis gp36 and E. chaffeensis gp47 were the predominantimmunoreactive proteins secreted into the supernatant (FIGS. 9A and 9B,lanes 1). The E. canis gp36 and E. chaffeensis gp47 were conclusivelyidentified in the supernate fractions with anti-recombinant gp36 andgp47 antibodies (FIGS. 9A and 9B, lanes 2). Supernatants from uninfectedDH82 cells did not contain any proteins recognized by antisera againstehlichiae (data not shown).

Example 9 Molecular Characterization of E. canis gp36 and E. chaffeensisgp47 Tandem Repeats Among Isolates from Different Geographic Locations

Concerning the major immunoreactive orthologous glycoproteins ofEhrlichia canis and E. chaffeensis, gp36 and gp47, the inventorscharacterized the tandem repeats molecularly. The genes encoding theseproteins contain tandem repeats near the carboxyl-terminus that aresites of O-linked glycosylation. Single repeat units from both gp36 andgp47 contain epitopes that are recognized by dog antisera but are notcross-reactive. Comparative analyses in limited numbers of NorthAmerican E. canis and E. chaffeensis determined that the tandem repeatsvaried in number and sequence among the isolates. To furthercharacterize the global conservation or heterogeneity of these proteins,particularly with respect to the tandem repeat regions, the gp36 andgp47 genes were amplified and compared in several continentallyseparated strains of E. canis and numerous geographically dispersedNorth American E. chaffeensis isolates. Primers were designed tointergenic regions upstream and downstream of the E. canis and E.chaffeensis gp36 and gp47 coding regions (E. canis gp36 forward 5′-AATCAA TGT AGT ATG TTT CTT TTA (SEQ ID NO:47) and reverse 5′-ATT TTA CAGGTT ATA TTT CAG TTA (SEQ ID NO:48); E. chaffeensis gp47 forward 5′-TTGTGC AGG GAA AGT TG (SEQ ID NO:49) and reverse 5′-AAT GAA AGT AAA TAA GAAAGT GTA (SEQ ID NO:50)), and amplification was carried out as previouslydescribed (Doyle et al., 2006) The E. canis gp36 gene sequences from theLouisiana (DQ146151; SEQ ID NO:52 polynucleotide encoding SEQ ID NO:53),Florida (DQ146152; SEQ ID NO:54 polynucleotide encoding SEQ ID NO:55),DJ (North Carolina)(DQ146153; SEQ ID NO:56 polynucleotide encoding SEQID NO:57), Sao Paulo (DQ146154; SEQ ID NO:58 polynucleotide encoding SEQID NO:59), and Cameroon 71 (DQ146155; SEQ ID NO:60 polynucleotideencoding SEQ ID NO:61) isolates and E. chaffeensis gp47 gene sequencesfrom the Jax (DQ146156; SEQ ID NO:62 polynucleotide encoding SEQ IDNO:63), St. Vincent (DQ146157; SEQ ID NO:64 polynucleotide encoding SEQID NO:65), V3 (Vanderbilt) (DQ146158; SEQ ID NO:66 polynucleotideencoding SEQ ID NO:67) and V8 (DQ146159; SEQ ID NO:68 polynucleotideencoding SEQ ID NO:69) isolates were deposited into the publiclyavailable GenBank database of the National Center for BiotechnologyInformation world wide website. The E. canis gp36 gene sequences fromthe Jake (DQ085427), Oklahoma (DQ085428), and Demon (DQ085429) isolatesand E. chaffeensis gp47 gene sequences from the Arkansas (DQ085430) andSapulpa (DQ085431) isolates were previously deposited. Sequence analysiswas performed as previously described (Doyle et al., 2006)

The tandem repeat sequence from North American, Brazilian, andCameroonian E. canis isolates was completely conserved, comprising nineamino acids (TEDSVSAPA; SEQ ID NO:22). However, the number of repeatsvaried between 4 and 18 copies of the repeat (see Table 4). TheN-terminal pre-repeat region (143 amino acids) was highly conservedamong all isolates, with the Jake, Oklahoma, Demon, and Louisianastrains containing complete homology. The Brazil and Cameroon isolatescontained the most divergent sequences (four differences in aminoacids), but these still retained 97.2% amino acid homology with theconsensus.

TABLE 4 Tandem repeats of gp36 and gp47 in different E. canis and E.chaffeensis strains Repeat Percent Tandem Repeat Strain number homologyE. canis gp36 Jake 12.2 100 Oklahoma 5.2 100 TEDSVSAPA (9 amino Demon16.2 99 acids) (SEQ ID NO: 22) Louisiana 5.2 99 Florida 4.2 100 DJ 18.2100 Sao Paulo 18.2 100 Cameroon 71 16.2 100 E. chaffeensis gp47ASVSEGDAVVNAVSQETPA Arkansas 7.0 99 (19 amino acids; SEQ ID NO: 23)EGNASEPVVSQEAAPVSE Jax 4.5 98 SGDAANPVSSENAS St Vincent 3.4 98 (33 aminoacids; SEQ ID Sapula 4.5 99 NO: 24) V3 4.5 97 V8 4.5 98

Similarly, the E. chaffeensis isolates tested demonstrated limiteddiversity of gp47 tandem repeats. The Arkansas strain exhibited a unique19 amino acid repeat unit compared to all of the other isolatessequenced. Seven repeats of the 19 amino acid sequence were identifiedin the Arkansas strain gp47 gene. Five additional E. chaffeensisisolates from geographically dispersed locations had a conserved 33amino acid repeat unit that exhibited minor variability in repeatsequence (see Table 4). The number of copies among this repeat wasidentical among three of four isolates (4.5 repeats), with the St.Vincent containing one fewer repeat (see Table 4). The N-terminal repeatpre-repeat regions (154 amino acids) were completely conserved betweenthe Sapulpa, St. Vincent, V3, and V8 strains. This sequence contained99.4% amino acid (one alteration) conservation with the pre-repeatregion of the Arkansas strain. More divergence was found in the Jaxstrain, with 91.6% amino acid homology (13 substitutions) in thepre-repeat region compared with the rest of the strains. Although theseproteins are highly immunoreactive and tandem repeat units of gp36 andgp47 contain the B cell epitopes (Doyle et al. 2006), interestingly, thelack of sequence divergence indicates that there is little immuneselective pressure on these proteins to alter their sequence or theymust be conserved to retain function, in specific embodiments.Similarly, comparative sequence analysis failed to detect significantsimilarity between the repeat units, demonstrating the tandem repeatsare not derived from common sequence that went through multiplemutations to gain diversity. The distinct tandem repeats of gp47 willfurther assist in differentiating variant strains into clades, and inparticular aspects of the invention provides insight into pathogenicdifferences among the strains. As these orthologous glycoproteins arehighly immunogenic (Doyle et al., 2006), the complete conservation oftandem repeats between strains of E. canis around the world and limiteddivergence between E. chaffeensis could have positive implications forfuture use of these proteins. With the tandem repeat units containingepitopes, a sensitive immunodiagnostic assay for E. canis is developedusing a recombinant protein with these tandem repeats.

Example 10 Vaccines of the Invention

In particular aspects of the invention, the immunogenic compositions ofthe present invention are suitable as a vaccine, such as a subunitvaccine. In other aspects of the invention, the immunogenic compositionsare referred to as immunoprotective.

Specifically, one or more compositions of the invention, such as thosecomprising an E. canis gp36 epitope or an E. chaffeensis gp47 epitope,for example, are administered to a mammal, such as a canine. Serum fromthe mammal may be assayed for an immune response, such as by detectingantibodies in the serum. The mammal is then subjected to subsequentchallenge with the pathogenic organism, such as the respective E. canisor E. chaffeensis organisms, and immunoprotection is determined.Controls may be employed, such as immunization with, for example, amutated epitope or an epitope that does not comprise a carbohydratemoiety. Complete or partial protection against the subsequent challengedemonstrates the immunoprotective nature of the composition, and thecomposition is a vaccine. Partial protection may be defined asprotecting from developing at least one symptom of the infection orprotecting from at least one symptom becoming worse.

Example 11 Significance of the Present Invention

The present inventors' previous study of the kinetic antibody responsesto E. canis revealed two major immunoreactive antigens (36- and 19-kDproteins) as dominant targets of the early host immune response (McBrideet al., 2003). The antigenic composition of E. canis in the tick is notknown, but antigens recognized early in the host immune response provideevidence of those that may be especially important in the initial stagesof infection of the mammalian host, and thus are high priority targetsfor molecular identification and for vaccine development. In thisinvention, the present inventors have conclusively identified andmolecularly characterized the E. canis 36 kD major immunoreactiveprotein, and, similar to several other major ehrlichial immunoreactiveproteins, it is glycosylated. In addition, a divergent and antigenicallydistinct 47-kD ortholog in E. chaffeensis, also a major immunoreactiveprotein consistently recognized by antibodies from HME patients, wasidentified. Other major immunoreactive proteins that have beenmolecularly characterized in E. canis include three glycoproteins(gp200, gp140, and p28/p30), and the identification of the gp36 in E.canis further supports glycosylation as an important ehrlichialpost-translational modification on several surface exposed proteins.

The E. canis gp36 and E. chaffeensis gp47 have considerable nucleic acidand amino acid divergence in regions containing theserine/threonine-rich tandem repeats. The recent genome sequenceanalysis of Ehrlichia ruminantium (Collins et al., 2005) and E. canis(unpublished data) has identified a high frequency of genes containingtandem repeat units. The E. canis gp140 and the E. chaffeensis gp120orthologs also have longer but genetically divergent, tandem repeats.None of the known glycoprotein repeat regions share conserved sequencesamong them, but all exhibit high serine and threonine content inaddition to alanine, proline, valine, and glutamic acid residues thathave been reported to serve as recognition motifs for 0-glycanattachment (O'Connell et al., 1991; Thanka Christlet and Veluraja,2001). The repeat units of the “mucin-like” glycoproteins were the onlylocation of predicted 0-glycan attachment as predicted by NetOGlyc, butnot all of the serines and threonines crossed the threshold as probablesites of glycosylation. However, as NetOGlyc was developed with datafrom eukaryotic glycoproteins, it is quite possible that the thresholdfor ehrlichial glycosylation is lower and that these are sites ofglycosylation. As with other known ehrlichial glycoprotein orthologs,the gp36 and gp47 are antigenically divergent. Consistentimmunodominance and divergence of the tandem repeats suggest that theimmune response creates strong selective pressures to alter the sequenceof the repeats. However, all E. canis strains tested contained identicaltandem repeats, but had variable repeat numbers (5 to 16). E.chaffeensis exhibited more divergence (amino acid sequence and repeatnumber) in the tandem repeat regions from two isolates that wereexamined (Arkansas and Sapulpa). Three of ten HME patient sera testeddid not react with the gp47 from E. chaffeensis Arkansas strain, and thedivergence in the repeat region of could explain the inconsistency ofgp47 recognition by different patients. A search for orthologous tandemrepeat DNA sequences throughout the genome does not detect pseudogenesor other sources for the nascent repeats, so the mechanism for repeatdivergence remains elusive. The discovery of a new pair of surfaceexpressed orthologs with repeat units is a point of interest in anobligate intracellular organism that has undergone reductive genomeevolution, and thus leads to speculation that there may be a selectiveadvantage to increase and retain the glycosylated repeat units of theseproteins.

Although the gp36 and gp47 have considerable homology in the N-terminalregions upstream of the repeat regions, the immunoreactive regions werelocalized to carboxyl-terminus region of the proteins, which containsthe tandem repeats. The present inventors determined that single repeatsfrom E. canis gp36 (9-mer) and E. chaffeensis gp47 (19-mer) expressed asrecombinant proteins were sufficient for antibody recognition by immunesera, demonstrating that they contain major repeated epitopes.Similarly, the repeat regions of the E. canis gp120 and E. chaffeensisgp140 contain major antibody epitopes. Interestingly, synthetic peptidesof the E. canis gp36 repeat (9-mer) and E. chaffeensis repeat (27-mer)were not recognized by immune serum and periodate treatment ofrecombinant repeat unit nearly abrogated antibody recognition, providingthe first evidence that the epitope determinants are complex and requirepost-translational modification for antibody recognition. In the absenceof organelles for protein trafficking, it was long believed thatprokaryotes did not contain the cellular machinery needed to modifyproteins with carbohydrates. Even E. coli, used for many years toexpress aglycosylated eukaryotic proteins, has been found to modify itsown proteins with carbohydrate moieties (Lindenthal and Elsinghorst,1999). Several human bacterial pathogens have now been discovered toexpress glycoproteins (Benz and Schmidt, 2002; Schmidt et al., 2003;Upreti et al., 2003) and the few prokaryotic glycoproteins functionallycharacterized contribute to adhesion, structural stability, andmobility, and are also targets of the immune system. Thesecharacteristics demonstrate the potential roles of bacterialglycoproteins in pathogenesis and immunity (Benz and Schmidt, 2002).Significantly, the carbohydrate-dependent antibody recognition ofrecombinant gp36 and gp47 expressed in E. coli demonstrates that glycansand attachment sites are conserved between native ehrlichial proteinsand recombinant glycoprotein expressed in E. coli. This indicates thatthe mechanisms for glycosylation are conserved between Ehrlichia and E.coli. However, glycosyltransferases homologous to those present in E.coli have not been identified in the E. canis genome (unpublished data),suggesting that these enzymes contain unique sequences and are among thehypothetical proteins with unknown function. Based on the identicalprotein masses and the dependence of post-translational modification forglycoprotein epitope reactivity, the recombinant ehrlichialglycoproteins appear to be identical to the native proteins in structureand composition, and thus are appropriate surrogates for native proteinsin studies to determine function and role as immunoprotective antigens.

The present inventors have observed that the gp36 and gp47 are presentin relatively low abundance in whole cell lysates compared to otherouter membrane proteins such as p28/p30. Several ehrlichial proteinshave been identified outside the bacterial cell including the gp120 andferric-ion binding protein. Furthermore, the E. chaffeensis gp120 hasbeen demonstrated to be differentially expressed on the surface ofdense-cored E. chaffeensis and extracellularly in the morula matrix.Immunogold electron microscopy demonstrated that E. canis gp36 and E.chaffeensis gp47 are also differentially expressed on the surface ofdense-cored ehrlichiae. The dense-cored and reticulate cellmorphological forms of Ehrlichia are thought to be homologous to theinfectious elementary body form of Chlamydia trachomatis andmetabolically active reticulate body, respectively. This observationindicates that these orthologous glycoproteins may play an importantrole in ehrlichial infection. The cell surface expression of theseglycoproteins indicates that they function as adhesins, in specificembodiments of the invention. Carbohydrate-lectin interactions arecommon means of bacterial adhesion, and E. chaffeensis has beendemonstrated to use L- and E-selectins to mediate cellular binding(Zhang et al., 2003). Repeat-containing proteins from Anaplasmamarginate as well as the “mucin-like” protein ortholog from Ehrlichiaruminantium have been demonstrated to confer to ability to adhere totick cells (de la Fuente et al., 2004).

The gp36 and gp47 are minor constituents in whole cell lysates, but thepresent inventors found substantial amounts of both in supernatants ofinfected cells. Interestingly, these were the abundant immunoreactiveproteins found in the supernatants in which other known surface proteinssuch as the p28/p30 were not detected, indicating that the gp36 wasindeed secreted and was not associated with intact outer membranes inthe supernatants. The secretion of gp36 in the tick salivary gland or inthe mammalian host would provide a partial explanation for the earlyhost immune response to this glycoprotein. Furthermore, secretion ofthese glycoprotein orthologs (gp36 and gp47) suggests that they may bevirulence factors and play an important role in pathobiology. A signalsequence was identified on the gp36, suggesting the involvement of a secdependent secretion mechanism such as Type II or Type IV (Nagai and Roy,2003). Genes encoding Type IV secretion machinery have been identifiedin E. canis, E. chaffeensis, and E. ruminantium (Collins et al., 2005;Felek et al., 2003; Ohashi et al., 2002) as the role of type IVsecretion of bacterial virulence factors is becoming better recognized.

The early immune recognition of gp36 by the mammalian host immuneresponse indicates the possibility that this antigen plays an importantrole in ehrlichial infection of the tick or in transmission and earlystages of infection, in specific embodiments of the invention. Althoughvery little is known with regard to ehrichial antigen expression in thetick, differential expression of Borrelia burgdorferi outer surfaceproteins has been demonstrated in the tick and restriction of A.marginate msp2 variants in ticks has been reported (Rurangirwa et al.,1999; Schwan and Hinnebusch, 1998). Successful infection of the ticks ormammalian host may be determined by the expression of specific outermembrane proteins required for host cell attachment or those involved inestablishment of intracellular infection.

Kinetics of the antibody response and antibody reactivity of the E.canis gp36 indicates that this antigen is useful in vaccine andimmunodiagnostic development. Current commercially available diagnosticassays for canine ehrlichiosis are based on p28/p30 proteins, and thegp36 could provide substantially better sensitivity for thisapplication. Furthermore, the gp36 antigen did not react with immunesera from E. chaffeensis dogs, which could be useful in developingspecies-specific immunodiagnostic assays. Similar serologic speciesspecificity has been reported with the gp120/gp140 as well as the gp200orthologs of E. canis and E. chaffeensis. These observations indicatethat the serologic cross-reactivity reported between E. canis and E.chaffeensis is not elicited by these major immunoreactive antigens. Thisfinding also has relevance to subunit vaccine development, indicatingthat these antigens are effective against homologous, in specificaspects of the invention. Vaccines that could potentially blockinfection during transmission would preferably contain antigensexpressed in the tick, and thus in specific embodiments the expressionof gp36 in the tick vector is determined.

The discovery of another set of major immunoreactive glycoproteinorthologs from ehrlichiae demonstrates their importance as targets ofthe immune system and their utility as immunoprotective antigens.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

PATENTS AND PATENT APPLICATIONS

U.S. Pat. No. 5,681,947

U.S. Pat. No. 5,652,099

U.S. Pat. No. 5,763,167

U.S. Pat. No. 5,614,617

U.S. Pat. No. 5,670,663

U.S. Pat. No. 5,872,232

U.S. Pat. No. 5,859,221

U.S. Pat. No. 5,446,137

U.S. Pat. No. 5,886,165

U.S. Pat. No. 5,714,606

U.S. Pat. No. 5,672,697

U.S. Pat. No. 5,466,786

U.S. Pat. No. 5,792,847

U.S. Pat. No. 5,223,618

U.S. Pat. No. 5,470,967

U.S. Pat. No. 5,378,825

U.S. Pat. No. 5,777,092

U.S. Pat. No. 5,623,070

U.S. Pat. No. 5,610,289

U.S. Pat. No. 5,602,240

U.S. Pat. No. 5,858,988

U.S. Pat. No. 5,214,136

U.S. Pat. No. 5,700,922

U.S. Pat. No. 5,708,154

U.S. Pat. No. 5,786,461

U.S. Pat. No. 5,891,625

U.S. Pat. No. 5,773,571

U.S. Pat. No. 5,766,855

U.S. Pat. No. 5,736,336

U.S. Pat. No. 5,719,262

U.S. Pat. No. 5,714,331

U.S. Pat. No. 5,539,082

U.S. Pat. No. 5,766,855

U.S. Pat. No. 5,719,262

U.S. Pat. No. 5,714,331

U.S. Pat. No. 5,736,336

WO 92/20702

PUBLICATIONS

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1.-35. (canceled)
 36. A method of identifying an E. canis or E.chaffeensis infection using a sample from an individual suspected ofhaving such an infection, comprising assaying the sample for an antibodythat immunologically binds a polypeptide consisting of: (a) a sequenceselected from the group consisting of SEQ ID NO:45 and SEQ ID NO:22; or(b) a sequence selected from the group consisting of SEQ ID NO:25, SEQID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:23 and SEQ ID NO:24wherein the sample is assayed by means of an immunoassay in whichantibodies from the sample, if present, will bind to an immobilizedpolypeptide comprising the sequence of (a) or (b) or a sequence 90%identical thereto, and further wherein bound antibodies are detected bymeans of a detectable label.
 37. The method of claim 36, wherein theimmunoassay is a enzyme linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay,chemiluminescent assay, bioluminescent assay, or a Western blot.
 38. Themethod of claim 36, wherein the antibody immunologically binds apolypeptide consisting of SEQ ID NO:45 or SEQ ID NO:22.
 39. The methodof claim 38, wherein the sample is assayed by contacting an immobilizedpolypeptide comprising a sequence selected from the group consisting ofSEQ ID NO:45 and SEQ ID NO:22, or a sequence 90% identical thereto, withsaid sample and detecting immunological binding of the antibody thereto.40. The method of claim 38, wherein the antibody immunologically binds apolypeptide consisting of comprising SEQ ID NO:22.
 41. The method ofclaim 40, wherein the sample is assayed by contacting an immobilizedpolypeptide comprising SEQ ID NO: 22 or a sequence 90% identicalthereto, with said sample and detecting immunological binding of theantibody thereto.
 42. The method of claim 36, wherein the antibodyimmunologically binds a polypeptide consisting of SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:23 or SEQ ID NO:24.
 43. Themethod of claim 42, wherein the sample is assayed by contacting animmobilized polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:23 and SEQ ID NO:24, with said sample and detectingimmunological binding of the antibody thereto.
 44. The method of claim36, wherein the antibody immunologically binds a polypeptide consistingof SEQ ID NO:23 or SEQ ID NO:24.
 45. The method of claim 44, wherein thesample is assayed by contacting an immobilized polypeptide comprisingSEQ ID NO:23 or SEQ ID NO:24, or a sequence 90% identical thereto, withsaid sample and detecting immunological binding of the antibody thereto.